Biofilm Formation of S.maltophilia with Vancomycin

Today I am repeating the crystal violet assay with several modifications to the staining procedure. In attempt to reduce the variability between trials, I will be using a different method to wash the plates after 24 hours of incubation. I will only be using three of our isolates of S.maltophilia (BAA2423, BAA84, and 13270) with vanc25 and vanc100, with no vanc as a control.  I have updated the protocol below to accommodate for the new wash method.

Materials

• Trypticase soy broth
• Cultures of bacteria on plates
• 15ml conical tubes
• Pipet aid with 5ml sterile glass pipet
• Shaker, 37°C
• 96 well plate, sterile
• Vacomycin stock
• Sterile distilled water
• 0.1% crystal violet
• p200 pipet and tips
• 95% ethanol
• 96 well plate reader

Procedure

1. Create an overnight culture of selected bacteria strains by adding 3ml TBS into each 15ml conical tube, add a loop of bacteria, and vortex well until bacteria is thoroughly suspended.
2. Place tubes in 37°C shaking incubator overnight.
3. Add 3mls of 2xTSB into new 15ml conical tubes labeled appropriately. From overnight cultures, pipet 30µl from culture into new media to create a 1:100 dilution.
4. Create a microtiter plate with different concentrations of vancomycin so that each strain will have a total of 18 wells (3 concentrations with 6 replicated wells).
• Create a 50μg/mL and 200μg/mL working stock
• Add 100μL to the wells that half of that vanc concentration
• Add 100μL of sterile water to all other wells
• Concentrations of vancomycin should be 0, 50, and 200 μg/ml.
5. Add 100µl of inoculated media into each designated well, final volume of all wells should be 200μL and final vanco concentrations will be 0, 25, and 100 μg/ml
6. Incubate plates at 37°C for 24 hours.
7. Carefully transfer culture from each inoculated well into a new 96-well plate and measure OD600 by using a plate reader set to 600nm.
8. Gently submerge the original plate in a 2 inch deep pan containing 0.1% crystal violet.
9. Let plate sit in the crystal violet for 15 minutes at room temp.
10. Remove the plate and gently submerge in a new pan containing distilled water.
11. Repeat in clean water 3 times to remove excess crystal violet and any unattached cells.
12. Let the plate dry for 15 minutes in the hood.
13. Add 200µl 95% Ethanol to each well, let sit for at least 5 minutes; vortex gently if necessary.
14. Read plate at 600 nm with plate reader.

//BEE 

Cockroach survival assay with Stenotrophomonas maltophilia and vancomycin

Today I am repeating the cockroach survival assay with S.malt. Instead of strains G and K, I will be using 13270 and the E.coli strain DH5alpha along with BAA84 and BAA2423 from liquid cultures I prepared yesterday.  I updated the protocol accordingly. Also, I placed the roaches in the 37C incubator 24 hours before injections so the roached will be better accumulated to incubation post injection. The purpose of this repeat is to see if we obtain similar results to what was observed in the first trial. In addition to the previous method, we added a titer plate method for calculating concentration of bacteria given in each injection to better understand results.

Protocol for Cockroach Survival Assay 

Materials 

• Bacterial cultures  
• Stenotrophomonas maltophilia isolates BAA 2423, BAA 84, and 13270 
• Escherichia coli DH5alpha  
• LB broth media  
• LB + Vancomycin100 broth media 
• 90 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 3 ml of LB broth in a sterile 15mL centrifuge tube.  Suspend a colony of the bacteria in the media and place in a 37°C shaking incubator for ~16 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 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. 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. 
6. Create control group by injecting 10 roaches with 25μL LB+Vancomycin100. 
7. Place 3-4 roaches of the same group into petri plates and store in 37°C incubator. Observe for survival for one week.
8. Create a titer for each strain by serial diluting  cultures 1:10 to the -8 dilution.
• This can be done by filling 8 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 -5 to -8 dilutions, spread 100μL onto an LB plate and place in 37°C incubator overnight. (Final dilutions with therefore be -6 to -9)
• Count colonies 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         9           9          9           9          9           9           90%

BAA2423                      9          8           8          7          7           7           7           70%

BAA2423+Vanco          3          3          2           2          2           1          1           10%

BAA84                          8          8           8          7          7           7           7           70%

BAA 84+Vanco            2           2           3          2          2           2          1           10%

13270                            2          1           1          1           1          1           1           10%

13270+Vanco                2          2           2          2          2           2           2           20%

DH5 alpha**                8          5           5           4          2          2           2            20%
DH5 alpha**+Vanco   10         5           2          1           1          1           1           10%

CFU/25μL (dosage per roach):
BAA 2423    3.7x10^7
BAA 84        1.6x10^7
13270          2.4x10^7
DH5 alpha   7.4x10^6

*During time of injections, the roaches seemed much healthier than the previous trial--may result in different immune response. 
**We are not confident in the identity of this strain of E.coli. Through various results we believe this strain is not DH5alpha, and therefore the study will be repeated using a different strain of either E.coli B/r or Klebsiella aerogenes. Furthermore, the study will be repeated with a lower concentraion of the same S.malt strians in order to observe results at a 1:10 dilution. 

//BEE 

Biofilm Formation of S.maltophilia with Vancomycin

Today I made cultures in TSB to test our 8 strains of S.malt with vancomycin to observe biofilm formation in a 96-well plate. I am repeating this study to compare results of previous trials to confirm similar results of an enhancement of biofilm at sub-inhibitory concentrations of antibiotic. I will be using the same protocol with crystal violet as I have outlined previously.

Results:

A significant increase in biofilm formation by isolates BAA 84, 13270, and 51 were observed at concentrations of vancomycin over 50 microgram/ml. This is consistent with previous trial, although we have seen a greater % increase in isolate 51 previously (>250%).

//BEE 

Biofilm Formation of S.maltophilia with Vancomycin

Today I am repeating an experiment I have done in the past that observers the effects the antibiotic vancomycin has on formation of biofilm on a polystyrene 96-well plate by the bacterium S.maltophilia. I started overnight liquid cultures yesterday of 8 ATCC strains that we have in the lab (BAA84, BAA2423, 700, 51, 49, 13270, 17666, 13637).  I followed the following procedure and will finish after 24 hours of incubation.

Materials

• Trypticase soy broth
• Cultures of bacteria on plates
• 15ml conical tubes
• Pipet aid with 5ml sterile glass pipet
• Shaker, 37°C
• 96 well plate, sterile
• Vacomycin stock
• PBS
• 0.1% crystal violet
• p200 pipet and tips
• 95% ethanol
• 96 well plate reader

Procedure

1. Create an overnight culture of selected bacteria strains by adding 3ml TBS into each 15ml conical tube, add a loop of bacteria, and vortex well until bacteria is thoroughly suspended.
2. Place tubes in 37°C shaking incubator overnight.
3. Add 3mls of 2xTSB into new 15ml conical tubes labeled appropriately. From overnight cultures, pipet 30µl from culture into new media to create a 1:100 dilution.
4. Create 2 microtiter plates with 4 different concentrations of vancomycin so that each strain will have a total of 16 wells (4 concentrations with 4 replicated wells).
• Create 200μg/mL working stock
• Add 200μL to the wells that will have the highest vanco concentration
• Add 100μL of sterile water to all other wells.
• Serial dilute 1:2 subsequent wells by transferring 100μL and discard final 100μL so all wells have a final volume of 100μL
• Concentrations of vancomycin should be 0, 50, 100, and 200 μg/ml.
5. Add 100µl of inoculated media into each designated well, final volume of all wells should be 200μL and final vanco concentrations will be 0, 25, 50, and 100 μg/ml
6. Incubate plates at 37°C for 24 hours.
7. Transfer culture from each inoculated well into a new 96-well plate and measure OD600 by using a plate reader set to 600nm.
8. Wash overnight plate 3 times with 200µl PBS per well, let dry for 30 minutes in the hood.
9. Stain wells with 200µl 0.1% crystal violet for 15 minutes.
10. Remove crystal violet and dispose of appropriately.
11. Wash wells 3 times with 200µl PBS, let dry for 15 minutes in the hood.
12. Add 200µl 95% Ethanol to each well, let sit for at least 5 minutes; vortex gently if necessary.
13. Read plate at 600 nm with plate reader.

//BEE 

Biofilm formation of S.maltophilia with delayed addition of vancomycin

Today I will be testing the impact on biofilm formation by S. maltophilia when vancomycin is added 6 hours after inoculation of a 96-well plate. I prepared liquid cultures yesterday in 3mLs of TSB and followed the procedure outlined below.

Materials

• Trypticase soy broth
• Cultures of 8 ATCC S. maltophilia bacteria isolates
• 15ml conical tubes
• Pipet aid with sterile pipets
• 37°C shaking incubator
• 37°C shaking incubator
• 96 well plate, sterile
• Vancomycin stock
• PBS
• 0.1% crystal violet
• p200 pipet and tips
• 95% ethanol
• 96 well plate reader

Procedure

1. Create an overnight culture of selected bacteria strains by adding 3ml TBS into each 15ml conical tube, add a loop of bacteria, and vortex well until bacteria is thoroughly suspended.
2. Place tubes in 37°C shaking incubator overnight.
3. Add 3mls of selected media into new 15ml conical tubes labeled appropriately. From overnight cultures, pipet 30µl from culture into new media to create a 1:100 dilution.
4. Add 200µl of inoculated media into each designated well
• For each isolate fill a 4x4 area for a total of 16 wells
5. Incubate plates at 37°C for 6 hours.
6. Remove liquid media from wells very carefully
7. Add 200µl of fresh TSB with to each row of 4 wells, with each column having a different concentration of vancomycin (0, 25, 50, 100µg/ml) and incubate at 37°C for 18 hours.
8. Remove liquid culture media from wells and transfer to a new 96 well plate--read OD600.
9. Wash overnight plate 3 times with 200µl PBS, let dry for 30 minutes in the hood.
10. Stain with 200µl 0.1% crystal violet for 15 minutes.
11. Remove crystal violet and dispose of appropriately.
12. Wash wells 3 times with 200µl PBS, let dry for 15 minutes in the hood.
13. Add 200µl 95% Ethanol to each well, let sit for 5 minutes.
14. Read plate at 600 nm with plate reader.

Tomorrow I will continue the procedure at step 8.

//BEE 

Minimal Inhibitory Concentrations of Vancomycin on S. maltophilia

Yesterday I read the 96-well plates that were used in the following protocol to determine the MIC of vacomycin on S.maltophilia. 

Minimal Inhibitory Concentration Determination Protocol

Materials

• Cultures of 20 Stenotrophomonas maltophilia isolates on plates
• 20 sterile 15mL centrifuge tubes
• 20 sterile 5mL Culture tubes
• 10 sterile 96-wells plates
• Sterile water (50mL)
• Tryptic soy broth (60mL)
• 2X tryptic soy broth (120mL)
• Working stock of 300 and 400μg/ml Vancomycin (16 mL each)
• 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. Make overnight liquid cultures from fresh plates in 3 mL TSB broth in 15 mL centrifuge tubes. Place in shaking 37°C incubator.
2. From overnight cultures, make 1:100 dilutions by inoculating 30μL culture into 6mL of fresh 2X TSB using 15mL conical centrifuge tubes.
3. 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.
4. Add 100μL of each diluted bacterial culture to 4 sets of dilutions.
5. 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. 
6. All wells should have a volume of 200μL
7. Place 96 well plates in 37°C incubator overnight.
8. After 24 hours, check OD600 with spectrometer for the minimal inhibitory concentration of vancomycin for Stenotrophomonas maltophilia.

Results
Isolate           MIC (μg/mL)
700                  100
BAA 2423       100
BAA 84           200
D                     200
E                       50
F                       50
G                     100
H                      25
I                      >200
J                       25
K                     150
L                      50
M                     50
N                    100
O                    >200
13637              50
17666             100
13270              150
51                   >200
49                    150

//BEE

Cockroach survival assay with Stenotrophomonas maltophilia and vancomycin

The purpose of my experiment that I am working on today is to observe the toxicity of Stenotrophomonas maltophilia when the antibiotic vancomycin is introduced. We have seen an increase in biofilm formation at sub-inhibatory concentration of vancomycin in several S. maltophilia isolates, as well as increased cytotoxicity by the amoeba Dictyostelium discoideum.

I struck out four of our laboratory strains of S. maltophilia (BAA 2423, 13270, G, and K) onto LB plates a few days ago. I used these plates yesterday to make overnight cultures that I used today, as directed in the following protocol that I used today.

Protocol for Cockroach Survival Assay

Materials

• Bacterial cultures
• Stenotrophomonas maltophilia isolates BAA 2423, 13270, G, and K
• LB broth media
• LB + Vancomycin100 broth media
• 70 Cockroaches ~1 inch 
• Insulin Syringe with 31G needle
• Repeat micropipetter 
• 2 mL Eppendorf tubes
• p1000 Pipettes and tips

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.
2. Transfer 1mL of the overnight liquid culture into a 2mL eppendorf tube. 
3. Make 2 replicates of each isolate for +/- vancomycin
4. Centrifuge cultures (2.5 min at 10,000g) into a pellet
5. Discard supernatant and resuspend one pellet with 1mL LB broth and the other with 1mL LB + vancomycin100. 
6. Incubate at 37°C for 2 hours.
7. Vortex and load an insulin syringe with resuspended bacterial culture and inject 25μL into each roach in a set of 10. Repeat with each bacterial culture.
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 daily for survival.

I will check the roaches every 24 hours for one week and record any deaths*.


RESULTS (number of roaches alive out of 10)

Group                         Day1   Day2    Day3    Day4    Day5    Day6    Day7  %survival

Control (LB+Vanco)    10         9          9          9           9          9          9          90%

2423                               8          6         5           5          5           5          5          50%

2423+Vanco                  6          3         2           1          0           0          0          0%

13270                            4          0          0           0          0           0          0          0%

13270+Vanco               8          8          7           7          6           6          6          60%

G                                   2          2          2           2          2           2          2          20%

G+Vanco                      2          1          1           1          1           0          0          0%

K                                   3          0          0           0          0           0          0          0%

K+Vanco                      5          2          1           1          1           1          1          10%

 

*roaches seemed very dehydrated with very little hemolymph. Hemolymph from dead roaches after 24 hours of injection was plated onto LB agar plates and put in 37degC incubator overnight. Bacterial colonies were observed on all plates.  This was done by sterilizing the ventral thoracial-coxal joint area of hindleg with 70% isopropyl alcohol, and then piercing with a 16G1 1/2 gauge needle to remove hemolymph with a sterile inoculating loop.

//BEE