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