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

Dehydration of Roaches affecting Survival

(from 11/14/14 Bd injections of Ft LVS)

Water crystals to prevent dehydration

There was a sudden death in the PBS and non-injected controls after 6 days (12% survival PBS and 50% survival in non-injected). This be attributed to dehydration after a two week incubation at 37C with only dry dog food.

Therefore, I have updated the protocol for roach storage for future experiments. Water crystals will be used as a source of water and larger containers will be used that can hold all 8 roaches for each group and allow for gas exchange (figure shown).

New containers

One concern is the roaches will now be stored in the walk-in 37C incubator used by the department to accommodate for the larger sized containers. This incubator is high-traffic due to teaching labs and the building's exterior door not far down the hallway. There is also a light that is often left on within the incubator. With these precautions in mind, results will be analyzed closely.






//BEE

LVS titer plate on CHOC II agar after 48 hr

(from 11/14/14 Bd injections of Ft LVS)

There were several deaths in Francisella groups. Data will be recorded one my Onedrive file here.
After 24 hours the E.coli spot-titer plates could be read, and all the Francisella plates were read after 48 hours to determine the CFU/dose of each injection group.

E.coli DH5alpha titer plate on LB after 24hr

Roaches contained in petri plates

//BEE

LD50s of Francisella LVS in Blaptica dubia roaches

Today I started an experiment to assay the virulence of the attenuated live vaccine strain (LVS) of Francisella tularensis. I am using Blaptica dubia roaches as the host organism.
The following protocol was used on today:

CHOC II plate with LVS

1. Acclamate roaches (each 0.7-0.9g) to 37degC at least 7 days prior to injections.
2. 2 days before: Streak CHOC II agar plates with bacterial cultures of LVS, dsbA, dipA, iglC, amd deoB. Plates were inverted and incubated at 37degC. 
3. E.coli DH5alpha was inoculated in LB broth and incubated at 37degC at 225 rpm.
4. 1 day before: From E.coli broth culture, perform an isolation streak on LB plate-incubate at 37deg C.
5. Day of Injections: 
• Using a sterile loop, scrape plate to obtain a large amount of bacterial colonies.
• Suspend in 1ml of sterile PBS to create a bacterial suspension (view images for quantity reference).



Amount of LVS that was used

• Serial dilute 10-fold to 10^-7.
• Inject a set of 8 roaches for each dilution 10^-1 to 10^-5.Wipe roach with isopropyl alcohol before injection
• Using sharpened pipette tips, pierce the 2^nd to last segment of the abdomen on the dorsal side.
• Inject 20μl of bacterial solution.
• Place 4 roaches in a sterile plastic petri dish for storage.
• Repeat for each bacterial strain.
6. Create a control group by injecting 8 roaches with PBS in the same way.
7. Place 8 roaches in petri plates for non-injected control.
8. Make a titer for each strain by dividing CHOC II plates (LB for E. coli) into 4 quadrants, and spotting five 10μl drops of the 10^-4 to  10^-7 10-fold dilutions; make two replicate plates.
9. Store all roaches at 37°C for 10 days—record any deaths.



Bacterial suspension of LVS in PBS

10.  Calculate LD₅₀ of each strain using titer and survival of roaches.

Storage of roaches in petri plates

//BEE

From 11/4/14

I have been testing the effects of gentamicin on dubia roaches. Today, I gave each one another dose of the antibiotic solution in the same manner as the original administration. All 10 roaches are still alive.

Observations: Because each roach had an individual piece of dog food in its petri plate, the oral feeding group was less accepting to the sucrose solution compared to the original administration 3 days ago. With enough patience and technique, each roach was given the full 10 microliter dose.

//BEE

Gentamicin dosing of Dubia Roaches

Today I designed an experiment to observe the effects of gentamicin, a non-cell-permeable antibiotic, on the survival of Blaptica dubia roaches. I will be using this antibiotic in the in vivo growth curve of the LVS strain of Francisella to observe intracellular and extracellular growth. This assay is inspired by a similar experiment that uses Drosophila melanogaster flies found here.

This experiment will include two different methods of antibiotic administration--oral feeding and injection. Future experiments will include the administration of different antibiotics using these methods, so this experiment will offer as a preliminary trial for future work.

Procedure:

1. Solutions of gentamycin were made in PBS and a 50% sucrose solution with a concentration of 1mg/mL.
2. A group of 5 roaches (each 0.7-0.9g) were fed 10µl of the gentamicin in 50% sucrose.
3. A group of 5 roaches were injected with 10µl of the gentamicin in PBS on the 2^nd to last segment of the abdomen on the left dorsal side. Site of injection was cleaned with 70% alcohol before. 
4. Each roach was stored in individual small plastic petri plates in the dark 37degC incubator.
5. Roaches will be administered antibiotic with the same dosing method twice a week for two weeks. Survival will be checked daily. 

//BEE 

LD50s of Listeria monocytogenes in Blaptica dubia roaches

Last Friday (10/24/14) I started an experiment for the high-throughput study of Listeria monocytogenes virulence using Blaptica dubia roaches.  I had previously determined the LD50 values of several mutants of Listeria from Dr. Teresa Bergholz lab in Galleria mellonella (wax worms). I using the roaches for add information about the isolates and to further develop the roach model.

The following protocol was used on Friday.

1. 2 days before: Request cultures from Becky and streak E.coli DH5alpha on an LB plate
2. 1 day before: Create overnight (20 hour) cultures in BHI broth and DH5alpha in LB broth and incubate at 37deg C, shaking >225rmp
3. Day of Injections: Serial dilute 10-fold to 10^-7.
• Inject a set of 8 roaches for each dilution 10^-1 to 10^-5.Wipe roach with isopropyl alcohol before injection
• Using sharpened pipette tips, pierce the 2^nd to last segment of the abdomen on the dorsal side.
• Inject 20μl of bacterial solution.
• Place 4 roaches in a sterile plastic petri dish for storage.
4. Create a control group by injecting 8 roaches with PBS in the same way.
5. Make a titer by dividing a plate into 4 quadrants, and spotting five 20μl drops of each 10-fold dilution on BHI plates (LB for E. coli).
6. Store roaches at 37°C for 10 days—record any deaths.
7.  Calculate LD₅₀ of each strain using titer and survival of roaches.

Sharpening of extra-long gel loading tips.

Recording of roach survival

//BEE

Francisella LD50 in Blaptica dubia roaches

from 10/17/14

Unfortunately due to complications with the media, spot titer plates did not produce individual colonies to determine CFU/dose for the three Francisella strains being tested. Because of this, the LD50 cannot be determined.

Francisella plates

However, the spot plates for Burkholderia K56-2 strain and E.coli DH5alpha were able to be read, and the PBS control showed no signs of bacterial contamination.

E.coli, K56-2, and PBS plates

Roaches in 37 deg C incubator

The roaches are being tracked for survival and will be observed for 6 more days.











//BEE 

LD50 of Francisella in Wax Worms

In addition to finding the LD50 values Francisella mutants in the Dubia roaches, I have also been repeating the work done by Ben to find the LD50s in the Greater Wax moth. I used the same protocol that was used for finding the LD50s of Listeria for Dr. Bergholz (Listeria LD50 protocol using wax worms).

A few modifications were made to accommodate for the fastidious nature of Francisella, which will not grow well in broth cultures. Mueller-Hinton plates with 2% Isovitalex were used to make 48 hour plates of each Ft culture. A loop of bacteria was suspended in 1ml of sterile PBS to form a bacterial suspension, which was then diluted with PBS.

//BEE

Francisella LD50 in Blaptica dubia roaches

Today I started an experiment for the high-throughput study of Francisella tularensis virulence using Blaptica dubia roaches. The purpose of this study is to ultimately develop a novel insect model that can be more applicable to long term studies.

The following protocol was used today.

1. Request blinded strains from dawn on Modified Mueller Hinton plates with appropriate times.
2. Suspend a colony of each bacterial strain in 1mL PBS.
3. Serial dilute 10-fold to 10^-6.
• Inject a set of 10 roaches for each dilution 10⁰ to 10^-5.Wipe roach with isopropyl alcohol before injection
• Using sharpened pipette tips, pierce the 2^nd to last segment of the abdomen on the dorsal side.
• Inject 20μl of bacterial solution.
4. Create a control group by injecting 10 roaches with PBS in the same way.
5. Make a titer by dividing a plate into 4 quadrants, and spotting five 20μl drops of each 10-fold dilution on Mueller-Hinton plates with 2% isovitalex.
6. Store roaches at 37°C for 10 days—record any deaths.
7.  Calculate LD₅₀ of each strain using titer and survival of roaches.

//BEE 

Lab Work Update

For the past month I have been working with Dr. Teresa Bergholz's lab. They have supplied me with Listeria monocytogenes and several mutants strains, and I have tested their virulence in Galleria mellonella (wax worms) to determine the lethal dose 50% (LD50). The following protocol was used for the 8 isolates being tested. A total of three replicates were done to find the final LD50s.

Listeria monocytogenes LD50 Protocol

1. Obtain inoculated plates from Becky
2. Give to Dawn for blinding of overnight broth cultures in BHI media 
• Incubate at 37°C shaking for 20 hours 
3. Remove 1 mL of bacterial suspention into a 2.0 mL epp tube 
4. Wash cells to remove BHI media and resuspend with PBS 
• Centrifuge at 13,000g for 1 minute in 2.0mL epp tube, wash twice. 
5. Make serial 1:10 dilutions of each overnight culture to 10-7 using 2.0 mL epp tubes and PBS as diluent 
6. Create a spot plate titer on BHI plates to determine CFU/mL of each dilution 
• Using 5x10μL spots for -4 to -7 dilutions 
7. With a Insulin syringe, inject 30μL into each worm 
• Set of 10 wax worms for each dilution of each mutant 
8. Create a control group by injecting 10 worms with PBS 
9. Place worms in labeled petri plates and securely store them at 37°C for 9 days, observe and record day and number of deaths and pupate 
10. Calculate LD50 for each mutant of Listeria using F020_LD50Gm excel file

Data was entered into the excel file and stored on my OneDrive account (Listeria Survival Data in wax worms) and found the following data:

Isolate    Rep 1          Rep 2         Rep 3         Average         St Dev
TB7        6.53E+03    3.39E+06    1.12E+04    1.14E+06    1.95E+06
TB10      4.18E+04    1.30E+03    1.13E+04    1.81E+04    2.11E+04
TB18      2.74E+04    1.62E+04    1.16E+04    1.84E+04    8.13E+03
TB19      4.77E+04    2.47E+03    1.10E+04    2.04E+04    2.40E+04
TB25      2.66E+05    5.56E+04    1.24E+05    1.48E+05    1.07E+05
TB26      7.07E+04    2.62E+04    6.18E+04    5.29E+04    2.35E+04
TB27      1.13E+04    1.53E+03    6.83E+03    6.55E+03    4.89E+03
TB133    4.18E+04    7.42E+04    8.25E+04    6.62E+04    2.15E+04
DH5a     3.39E+06    6.60E+04    1.47E+05    9.79E+05    1.61E+06
                                   3.13E+05   

Table can be viewed in the excel file here:Listeria LD50 Values     

//BEE 

Update

For the past two weeks I have been testing the cytotoxicity of several ATCC S.maltophilia strains when the antibiotic vancomycin is present. I have been using the dictyostelium plaque assay to compare the PFU50 with and without vancomycin at a concentration of 25micrograms/ml. So far I have not seen a significant difference in PFU50 when vancomycin is present. The antibiotic stock was made fresh each time the plates were made and fresh bacterial cultures were always used.

It was decided that roaches should be acclimated for 3 weeks at 37 deg C before using for experiments, so I will start my next survival trial when I get back using Francisella and a few mutants to determine LD50 in the dubia roaches.

//BEE 

PFD50 with Dicty and Vancomycin

Today I am using the Dicty predation assay to observe the cytotoxicity of several strains of bacteria when vancomycin in present on the media. I will be using eight Stenotrophomonas maltophilia strains from the ATCC: BAA2423, BAA84, 700, 13270, 17660, 13637, 51, 49; Escherichia coli B/r; Klebsiella aerogenes; Burkholderia cenocepacia K56-2. The purpose of this experiment is to see if there is an increase in the cytotoxicity of the bacteria in the presence of vancomycin. If so, we will see an increase in the PFD50 (plaque forming unit) when vancomycin is present in the media. Klebsiella and E.coli will be used as positive controls. I will be using the following protocol.

Dictyostelium PFD₅₀ Protocol

Materials

• Cultures of selected bacteria
• S. malt BAA2423, BAA84, 700, 13270, 17660, 13637, 51, 49 
• Escherichia coli B/r 
• Burkholderia cenocepacia K56-2 
• Klebsiella aerogenes  
• Dictyostelium discoideum grown on E.coli B/r
• 2.0mL eppendorf tubes
• p200 pipette with tips
• p1000 pipette with tips
• Sterile loops
• Sterile spreaders
• Sterile water
• p20 multichannel pipette with tips
• 96-well plate
• SM/2 +Vanco²⁵ plates
• SM/2 plates

Protocol

1. Make overnight cultures of bacteria strains by suspending a colony in 3mLs of LB in a 15 mL centrifuge tube and place tubes in 37°C shaking incubator for ~16-20 hours
2. From overnight cultures, pipet 100µL of bacteria solution onto one SM/2+Vanco²⁵ plate and one SM/2 plate. Spread the solutions evenly with a sterile spreader to create a bacterial lawn and let dry in the hood for 30 minutes.
3. Pipette 1mL of sterile water into a 2.0mL eppendorf tube and suspend 4-5 dicty fruiting bodies.
4. Serial dilute tube 1:10 to 10^-7 in 2.0mL eppendorf tubes

• Add 900µL sterile water to seven 2.0mL tubes and transfer 100µL to the subsequent tube (change pipette tips in between transfers and mix each suspension thoroughly).
5. In a 96 well plate, fill one row with the 8 dilutions of Dictyostelium with 300µL, and refill the wells when needed.
6. With a multichannel pipet, allocate 4 rows of 2 µL of the dilutions onto each plate.
7. Let the plate sit upright for at least 30 minutes to prevent the spots from running tßogether.
8. Create a titer by pipetting 100µL of the overnight culture of Klebsiella onto 15 SM/2 plates.
9. Add 10 µL of the 10^-1 to 10^-6 dilution to a set of 3 plates and spread the liquid out evenly (3 replicates for an accurate titer).
10. Observe the plates every day for one week for plaque formation.
11. Before titer plates are overgrown, calculate the PFU for available plates.

//BEE 

MIC of Vancomycin with K56-2, E.coli B/r, and K.aerogenes

Today I am testing the resistance to the antibiotic vancomycin by 3 different bacteria strains. The purpose of this is to help understand results of upcoming roach survival assays with this antibiotic added to the three strains. Burkholderia cepacia K56-2, Escherichia coli B/r, Klebsiella aerogenes, and Esherichia coli DH5alpha (from a different source other than the one used before) will be used. Overnight liquid cultures were made in 10mL LB in 50mL centrifuge tubes from plates made from freezer stocks. The strains will be tested using the protocol used before and shown below.

Minimal Inhibitory Concentration Determination Protocol

Materials

• Bacterial cultures on plates
• Sterile 5mL Culture tubes
• 2 sterile 96-wells plates
• Sterile water
• Tryptic soy broth (60mL)
• 2X tryptic soy broth (120mL)
• Working stock of 300 and 400μg/ml Vancomycin
• Sterile inoculating loops
• Multichannel p200 pipette with sterile tips
• p20 pipette with sterile tips
• Sterile 50mL reservoirs
• 37°C shaking incubator
• 37°C stationary incubator
• Sterile 10mL pipettes with pipette aid

Procedure

1. From overnight cultures, make 1:100 dilutions by inoculating 60μL culture into 6mL of fresh 2X TSB using 15mL conical centrifuge tubes.
2. Create a microtiter plate with 12 different dilutions of vancomycin.
1. Create 300 and 400μg/mL working stock
2. Add 200μL of 400μg/mL to the wells that will have the highest vanco concentration
3. Add 100μL of 300μg/mL to the wells that will have a final concentration of 150μg/mL
4. Add 100μL of sterile water to all other wells.
5. Serial dilute 1:2 subsequent wells by transferring 100μL and discard final 100μL so all wells have a final volume of 100μL
6. Concentrations of vancomycin should be 0, 0.78, 1.562 , 3.125, 6.25, 12.5, 25, 50, 100, 200, 300 , 400 μg/ml.
3. Add 100μL of each diluted bacterial culture to 4 sets of dilutions.
4. The final concentrations of vancomycin should be 0, 0.39, 0.78, 1.562, 3.125, 6.25, 12.5, 25, 50, 100, 150, and 200 μg/ml. 
5. All wells should have a volume of 200μL
6. Place 96 well plates in 37°C incubator overnight.
7. After 24 hours, check for the minimal inhibitory concentration of vancomycin for each strain.

Results

E.coli DH5alpha    50μg/mL

E.coli B/r               50μg/mL

K. aerogenes         >2000μg/mL

B. cepacia K56-2  >200μg/mL

//BEE 

Cockroach survival assay with Stenotrophomonas maltophilia and vancomycin

Today I will be repeating the roach survival assay with several changes to the previous protocol.  I will be using a 1:10 dilution of the overnight culture I prepared yesterday to inject the roaches. We want to see if there will be similar results with a lower concentration of cells. I will also be using the Escherichia coli B/r strain and Klebsiella aerogenes in addition to the three S.maltophilia strains used previously (BAA84, BAA2423, and 13270).  I will be using the "drop plate" method for the titer plates because it has been recommended for more accurate results.

Protocol for Cockroach Survival Assay 

Materials 

• Bacterial cultures  
• Stenotrophomonas maltophilia isolates BAA 2423, BAA 84, and 13270 
• Escherichia coli B/r
• Klebsiella aerogenes
• LB broth media  
• LB + Vancomycin100 broth media 
• 150 Cockroaches ~1 inch  
• Insulin Syringe with 31G needle 
• Repeat micropipetter  
• 2 mL Eppendorf tubes 
• p1000 Pipettes and tips
• 37°C incubator (shaking and stationary)

Procedure 

1. From fresh plates of bacteria, make an overnight culture using 10 ml of LB broth in a sterile 50mL centrifuge tube.  Suspend a colony of the bacteria in the media and place in a 37°C shaking incubator for ~16-20 hours. 
2. Transfer 1mL of the overnight liquid culture into a 2mL eppendorf tube.  
• Make 2 replicates of each isolate for +/- vancomycin 
3. Spin down cultures (2.5 min at 10,000g) into a pellet 
4. Discard supernatant and resuspend one pellet with 1mL LB broth and the other with 1mL LB + Vancomycin100.  Repeat for each isolate.
5. Let cultures incubate for 2 hours at 37°C
6. Dilute all cultures 1:10 by transfering 100μL into 900μL of the same media and antibiotic concentration.
7. Load an insulin syringe with resuspended bacterial culture and inject 25μL into each roach in a set of 10 using a repeat micropipetter (set at 2.5 where 1 Div= .01 mL). Repeat with each bacterial culture +/-vancomycin. 
• Make sure roaches have been incubated at 37°C for at least 24 hours before injection. 
• For E.coli B/r and K.aerogenes, use both diluted and undiluted cultures because there is no data for undiluted survival.
8. Create control group by injecting 10 roaches with 25μL LB+Vancomycin100. 
9. Place 3-4 roaches of the same group into petri plates and store in 37°C incubator. Observe for survival for one week.
10. Create a titer for each strain by serial diluting  the final cultures 1:10 to the -7 dilution and using the drop plate method.

• This can be done by filling 7 1.5mL eppendorf tubes with 900μL sterile PBS and transferring 100μL to the subsequent tube (change pipette tips in between transfers and mix each suspension thoroughly). Repeat for each isolate used.
• From the -4 to -7 dilutions, pipette 5 spots of 10μL onto an LB plate divided into 4 quadrants, as shown by image. 
• Repeat for each dilution for each bacterial strain used. 
• Let plates sit upright until spots abosrb into plate.
• Invert and place in 37°C incubator overnight. (Final dilutions with be -6 to -9)
• Count colonies in the 3-30 range and determine CFU/mL for injection cultures. 

RESULTS (number of roaches alive out of 10)

Group                               Day1  Day2  Day3  Day4  Day5  Day6  Day7  %survival

Control (LB+Vanco)         10      10      10        10      10        10       10          100%

BAA2423                            10        7        7          5         5          5         5            50%

BAA2423+Vanco               8         6        5         4         4          4         4            40%

BAA84                                 8         5        4          4         3         2         2            20%

BAA 84+Vanco                  6         4        3          3         1          1          1            10%

13270                                   6         4        3          3         3         3         3            30%

13270+Vanco                     9         5        4          4         3          3         3            30%

E.coli B/r (10^-1)              9         3        2          2         2         2         2            20%
E.coli B/r+Van(10^-1)     10       6        5          3         2          2         2            20%
E.coli B/r (undilute)          2        1         1          1         1          0         0            0%
E.coli B/r+Van(undilute) 5        0        0         0         0          0         0            0%
K.aerogenes (10^-1)          4        4         4         4         4         4          4            40%
K.aer+Van (10^-1)             3        2        2          2         2         2          2            20% 
K.aer (undilute)                 2        2         2          2         2         2         2            20%
K.aer+Van(undilute)         1        1         1           1         1          1          1            10%

CFU/25μL (dosage per roach):
BAA 2423                           1.07x10^6
BAA 84                                1.35x10^6
13270                                  1.10x10^6
E.coli B/r (10^-1)            8.50x10^5
E.coli B/r (undilute)       8.50x10^6
K.aerogenes (10^-1)        3.00x10^5
K.aerogenes (undilute)   3.00x10^6

//BEE