Biofilm Assays on Fibrinogen-coated Silicone Catheters and 96-well Polystyrene Plates

Materials and Reagents

1. Bacterial growth and biofilm assay
   
   1. 500 ml Corning disposable sterile bottle-top filters with 0.22 µm Membrane (Fisher Scientific, catalog number: 09-761-112)
   2. 500 ml Reusable Glass Media Bottles with Cap (Fisher Scientific, catalog number: FB800500)
   3. 100 x 15 mm Petri Dish (Fisher Scientific, catalog number: S43570)
   4. 1 µl inoculating loops (DB Difco, catalog number: BD 220215)
   5. 15 ml Conical tube, for growing microaerophilic bacteria (Fisher Scientific, catalog number: 50-153-5104) 
   6. Disposable Round-Bottom rimless glass tubes (Fisher Scientific, catalog number: 14-962-15A) and cap (Fisher Scientific, catalog number: 14-957-91)
   7. Cuvettes, Standard: Polystyrene (Fisher Scientific, catalog number: 14-955-127)
   8. 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 02-682-002)
   9. Pipette tips
   10. pH strips (EMD Millipore, catalog number: 1.09542.0001)
   11. Catheter-Nalgene 50 silicone tubing (Nalgene Brand Products, catalog number: 80600030)
   12. 96-well polystyrene plates (Grenier Bio-One CellSTAR, catalog number: 655180)
   13. 96-well Microtitration plates (Corning, catalog number: 3788)
   14. Axygen Scientific microplate presterilized sealing tape (Fisher Scientific, catalog number: 14-222-044)
   15. Bacterial species: Enterococcus faecalis OG1RF (ATCC 47077)
   16. Double deionized water
   17. 1x Phosphate sodium saline (Sigma-Aldrich, catalog number: P3813)
   18. 0.1 N Hydrochloric acid solution (HCl) (Sigma-Aldrich, catalog number: 2104)
   19. 0.1 N Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: SX0607C)
   20. Bovine serum albumin (Sigma-Aldrich, catalog number: A7906)
   21. Agar, Bacteriological Grade (BD Difco, catalog number: B281230)
   22. Human fibrinogen free from plasminogen and von Willebrand factor (13-14 mg/ml–concentration varies with each batch)(Enzyme Research Laboratory, catalog number: FIB 3)
   23. Brain Heart Infusion broth (BHI) (BD Company, catalog number: B237500) or any other media for the requirements of your bacterial species 
   24. Pooled human urine (collected from at least three healthy female donors [Internal Review Board approval needed] or fresh urine commercially available).
       Note: Urine should be fresh and stored under refrigeration for no longer than 3 days.
   25. BHI liquid media (see Recipes)
   26. BHI-agar plates (see Recipes)
   27. Urine (see Recipes)
       
       
2. Assessment of biofilm formation on catheters
   
   1. 12-well polystyrene microplate (Fisher Scientific, catalog number: 08-772-50)
   2. Aluminum foil
   3. 10% Neutral Buffered Formalin (Fisher Scientific, catalog number: 22-046-361)
   4. 20% Sodium azide solution (Sigma-Aldrich, catalog number: S-2002)
   5. Tween-20 (Sigma-Aldrich, catalog number: P1379)
   6. Methyl α-d-mannopyronoside (Sigma-Aldrich, catalog number: M6882)
   7. Rabbit anti-Enterococcus antibody (Abcam, catalog number: ab68540) or any other anti-Enterococcus antibody commercially available
   8. IRDye 680LT goat anti-rabbit (LI-COR Biosciences, catalog number: 926-68021)
   9. Immunostaining Solutions (see Recipes)
      
      1. Blocking Solution
      2. Wash Solution
      3. Dilution Buffer
      4. Primary antibody solution
      5. Secondary antibody solution
   
   
3. Assessment of Biofilm on microplate
   
   1. Paper towel
   2. Reagent reservoir (Fisher Scientific, catalog number: 14-387-065)
   3. Crystal violet (Sigma-Aldrich, catalog number: C0775)
   4. Acetic acid (Sigma-Aldrich, catalog number: A6283)
   5. Solutions (see Recipes)
      
      1. 0.5% Crystal violet solution
      2. 33% of Acetic Acid

Equipment

1. Forceps
2. Heraeus Multifuge X3R Refrigerated Centrifuge (VWR, catalog number: 75004516) or any refrigerated centrifuge with similar features
3. Spectra Max ABS Plus Spectrophotometer (Molecular Devices, catalog number: ABS PLUS) or any spectrophotometer with similar features
4. Branson Ultrasonics^TM Bransonic^TM CPX-sonicator (waterbath sonicator) (Fisher Scientific, catalog number: 15-337-419) or any waterbath sonicator with similar features
5. Odyssey CLx imaging system (LI-COR, catalog number: 9140-01)
6. Test tube racks (Fisher Scientific, catalog number: 14-809-62) or any standard test tube racks
7. VWR Microbiological Incubator (VWR, catalog number: 51030017) or any standard microbiological incubator
8. Biological Safety Cabinet with UV light (Thermo Fisher, catalog number: 1395) or any standard equipment with similar features
9. Vortexer (Fisher brand, catalog number: 02-215-418) or any standard vortex
10. Autoclave (Getinge, catalog number: 633LS) or any standard autoclave
    

Software

1. CLX image studio (LI-COR, catalog number: 9140-510)
2. GraphPad Prism (GraphPad Software LLC)
   

Procedure

The individual steps of this protocol are summarized in Figure 1.


Figure 1. Protocol layout

1. Bacterial growth
   
   1. Streak the bacterial species on a BHI agar plate using a sterile inoculation loop.
   2. Incubate bacteria overnight at 37 °C.
   3. Add 10 ml of BHI media into a 15 ml conical tube.
   4. Using a sterile inoculation loop, pick a single E. faecalis colony to inoculate the media.
   5. Incubate the bacterial culture for 18 h at 37 °C under static conditions (target OD₆₀₀ = 1.0).
   
   
2. Catheter and microplate preparation
   Catheter
   
   1. Cut the silicone tubing in 1 cm pieces.
   2. Cut in half the 1 cm pieces (Figure 2).
      
      
      Figure 2. Preparation of 1 cm silicone pieces. A. Silicone tubing. B. 1 cm silicone pieces. C. 1 cm pieces in half.
      
      
   3. Put the resulting pieces in an open Petri dish.
   4. UV-sterilize the pieces overnight (Biological Safety Cabinet standard UV settings)
   5. Use sterile forceps to transfer each silicone piece into sterile 5 ml glass test tube and cap it.
      Note: Fibrinogen is a sticky protein. Therefore use of a glass tube will reduce binding of fibrinogen to the tube walls. 
   6. Thaw the fibrinogen stock solution at 37 °C, and bring the 1x PBS solution to 37 °C. Fibrinogen is soluble at body temperature; therefore, keep it at 37 °C until you add it to the silicone pieces. 
   7. Prepare a working solution of 100 µg/ml of fibrinogen in 1x PBS. 
   8. Add 1 ml of the fibrinogen solution to the test tube containing the silicone piece. 
   9. Incubate the silicone pieces at 4 °C overnight under static conditions to allow fibrinogen to coat the catheter.
      
      
   Microplate
   10. Dispense 100 µl of the 100 µg/ml fibrinogen solution (Step B7) into each well of the 96-well polystyrene plates (Grenier Bio-One CellSTAR). 
   11. Seal the plate with a sterile plate sealing tape.
   12. Incubate the microplate at 4 °C overnight to allow fibrinogen to coat the bottom of the well.
   
   
3. Culture preparation
   
   1. Centrifuge the overnight culture (Step A5) for 10 min at 7,000 rpm (7,505 x g).
   2. Remove supernatant.
   3. Resuspend the bacterial pellet with 10 ml of 1x PBS solution. Then centrifuge again for 10 min at 7,000 rpm (7,505 x g). Wash the bacterial cells by resuspending the bacterial pellet with 10 ml of 1x PBS solution. Repeat this step 3 times.
   4. Dilute 1 ml of bacterial solution into 9 ml of 1x PBS solution (dilution 1:10). This step is necessary to ensure the accuracy of the measurement.
   5. Take 1 ml of the diluted solution and put it into a cuvette. 
   6. Measure optical density (OD₆₀₀) by using the spectrophotometer.
      Note: Multiply the OD₆₀₀ value by 10 (dilution factor) to obtain the final optical density of the culture. 
   7. Dilute the culture to a final OD₆₀₀ of 1.0.
   8. Supplement fresh filter-sterilized urine (see Recipe 2) with 20 mg/ml of BSA. 
   9. Filter sterilize the supplemented urine using bottle top filter. 
   10. Inoculate 1:100 of normalized culture (Step C7) into the filter-sterilized urine supplemented with BSA. 
   
   
4. Catheter Fg-dependent biofilm setup
   
   1. After overnight incubation at 4 °C, remove the test tubes containing the Fg-coated silicone pieces (Step B9). 
   2. Aspirate the fibrinogen solution gently using a 1,000 µl pipette. 
   3. Add 1 ml of the bacteria-containing urine (Step C10). As negative control, incubate three Fg-coated pieces with only BSA-supplemented urine (no bacteria).
   4. Incubate the tubes under static conditions at 37 °C for 24 h (or as needed). 
   
   
5. Assessment of the catheter biofilm
   
   1. After overnight incubation at 4 °C, remove the Fg-coated plates (Step B12). 
   2. Peel off the plate sealing tape. 
   3. Aspirate the fibrinogen solution gently using a pipette.
   4. Add 200 µl of the bacteria-containing urine (Step C10). As negative control, incubate 8 Fg-coated wells (one column of the microplate) with only urine (no bacteria).
   5. Cover the plate with a sterile lid. 
   6. Incubate the microplate tubes under static conditions at 37 °C for 24 h (or as needed). 
   
   
6. Assessment of the microplate biofilm
   
   1. Aspirate the bacterial culture from the tube using a 1,000 µl pipette.
   2. Remove the unbound bacteria by pipetting vigorously using 1 ml of 1x PBS at room temperature. Repeat this step 3 times.
      
      
   Assessment of biofilm formation by colony forming units 
3. Transfer the silicone piece with sterile forceps (from Step F2) into a 15 ml conical tube. 
4. Add 1 ml of 1x PBS. 
5. To detach the bacterial biofilm from the catheter, vortex for 30 s (maximum vortex’s speed) at room temperature. 
6. Put conical tube into the Branson ultrasonic bath for 5 min at room temperature (40 kHz frequency), and vortex for another 30 s.
7. Take a 200 µl sample and serially dilute it with PBS (1:10).
8. Plate dilutions on BHI agar plates.
9. Incubate the plates at 37 °C overnight. 
10. Quantify colony forming units (Figure 3).
    
    
Assessment of biofilm formation by immunostaining
3. After washing the silicone pieces (Step F2), transfer them to a 12-well plate.
4. Fix the silicone pieces by adding 5 ml of 10% neutralizing formalin solution for 20 min.
5. Remove the formalin
6. Wash the pieces by adding 5 ml of PBS (repeat 3 times). All washes in this protocol are done under static conditions at room temperature unless otherwise noted.
7. Block the pieces by adding 5 ml blocking solution for 1 h at room temperature (or at 4 °C overnight).
8. Wash the pieces with 5 ml wash solution. Repeat this step 3 times.
9. Add 5 ml of the primary antibody solution. 
10. Incubate for 2 h at room temperature under static conditions.
11. Wash the pieces with 5 ml wash solution. Repeat this step 3 times.
12. Add 5 ml of the secondary antibody solution. Cover the plate with aluminum foil (secondary antibody is sensitive to light).
13. Incubate for 1 h at room temperature under static conditions
14. Wash the pieces with 5 ml wash solution. Repeat this step 3 times.
15. Transfer the pieces into a new 12-well plate.
16. Let them dry overnight.
17. Visualize the biofilm using the Odyssey imager (detection at 700 nm near-infrared region) (Figure 4).
    
    
Colorimetric assessment of biofilm formation by crystal violet 
3. After washing the silicone pieces (Step F2), transfer them to a 12-well plate.
4. Let pieces air dry. 
5. Add 5 ml of 0.5% of crystal violet solution.
6. Stain for 10 min under static conditions at room temperature.
7. Wash the pieces with distilled water (repeat this step 3 times).
8. Add 1 ml of 33% acetic acid to solubilize the crystal violet staining.
9. Incubate for 15 min at room temperature under static conditions.
10. Transfer 200 µl of the solubilized crystal violet into a new microplate.
11. Quantify absorbance at 595 nm using a plate reader. As a blank, use the 33% acetic acid in water. Serial dilution may be required to fall into the linear range of the plate reader. 


7. Assessment of the microplate biofilm
   

1. After incubation (Step E6), remove the culture by inverting the plate. 
2. Wash the unbound cells by submerging the plate in a small container full of water. Shake the plate gently and shake out the water. Repeat this step 3 times.
3. Dry the plate by tapping gently on a paper towel to remove residual liquid.
4. Let the plate air dry.
5. Add 200 µl of 0.5% of crystal violet solution.
6. Stain for 10 min under static conditions at room temperature.
7. Rinse by submerging the plate into the container full of water. Once submerged, shake the plate vigorously, dump the liquid, and tap the plate on a paper towel to remove the residual liquid. Repeat this step 3 times.
8. Add 200 µl of 33% acetic acid to solubilize the crystal violet staining.
9. Incubate for 15 min at room temperature under static conditions.
10. Transfer 200 µl of the solubilized crystal violet into a new microplate.
11. Quantify absorbance at 595 nm using a plate reader. As a blank use the 33% acetic acid in water. Serial dilution may be required to fall into the linear range of the plate reader.
    

Data analysis

1. Analysis of biofilm-associated CFU
   
   1. Log₁₀ transform final bacterial counts recovered from each catheter (n ≥ 3) (Figure 3).
   2. Plot data on a linear scale and include the arithmetic mean. 
   3. Depending on the amount of groups you want to compare, analyze the log₁₀-transformed data with either a two-tailed Mann-Whitney U test or a one-way ANOVA followed by an appropriate post-test comparison.
      Note: Log₁₀ transformation is optional. An alternative way to plot and analyze the data would be to plot untransformed data on a log scale with the geometric mean and the geometric standard deviation.
      
      
      Figure 3. Representative data of biofilm measured by colony forming units (CFU). Comparison of biofilm formation between E. faecalis OG1RF (wild-type strain) and a manganese uptake system triple mutant strain (ΔefaΔmntH1ΔmntH2), which is a biofilm deficient mutant. Two-tailed Mann-Whitney U tests were performed to determine significance between two groups (***P < 0.0002).
   
   
2. Analysis of biofilm formation assessed by immunostaining or crystal violet
   
   1. Plot the relative fluorescence to the negative control (Figure 4) or absorbance (relative to the negative control) for each group (n ≥ 3) on a linear scale and include the arithmetic mean and the standard deviation.
   2. Analyze the data with a Mann-Whitney U test.
      Notes:
      
      1. The biofilm formation assays on catheters often do not follow a normal distribution as determined by the D’Agostino-Pearson test. Thus, a nonparametric analysis such as the Mann-Whitney U test is more appropriate than the parametric ANOVA test. 
      2. Outliers can be identified using the ROUT method (Q = 1% recommended) and can be removed from the final analysis if confirmed.
         
      
      
      
      Figure 4. Representative images and data of biofilm analyzed by immunostaining and crystal violet. A. Visualization of biofilm formation on catheter pieces by E. faecalis OG1RF and a manganese uptake system triple mutant strain (ΔefaΔmntH1ΔmntH2). Catheter controls are those Fg-coated catheters that were incubated with only urine (no bacteria). B. Immunostaining analysis of biofilm formation between OG1RF WT and deficient mutant by plotting fluorescence relative to catheter controls. C. CV staining to quantify and compare biofilm formation between OG1RF WT and deficient mutant by plotting absorbance relative to catheter controls. Two-tailed Mann-Whitney U tests were performed to determine significance between two groups (***P < 0.0002).
      

Recipes

1. Media
   
   1. BHI liquid media
      Add 37 g of BHI broth powder into 1 L of distilled water
      Mix until solution is clear
      Autoclave for 20 min
      Let media cool down to room temperature prior to use
   2. BHI-agar plates
      Add 37 g of BHI broth powder and 15 g of agar into 1 L of distilled water
      Mix until solution is clear
      Autoclave for 20 min
      Let media cool down to a temperature of 45 °C
      Add antibiotics if needed and mix
      Keep media warm and pour 25 ml of media into Petri dishes
2. Urine
   
   1. Pool the urine from at least three healthy female donors (or get commercially available pooled urine). Use the same urine batch for consistency during the study
   2. Centrifuge pooled urine for 5 min at 7,000 rpm (7,505 x g) to remove precipitates
   3. Measure pH using pH strips and adjust to 6.5 using HCl solution or NaOH solutions as needed
   4. Filter sterilize the pooled urine using bottle top filters. Preferentially use fresh urine every time. In case, it is not used right away, store urine at 4 °C for no longer than 3 days. Repeat centrifugation for 5 min at 7,000 rpm (7,505 x g) if formation of precipitates is observed
3. Solutions
   
   1. Blocking Solution
      1x PBS with 1.5% BSA and 0.1% Sodium Azide. Keep solution at 4 °C
   2. Wash Solution
      1x PBS with 0.05% Tween-20. Keep solution at room temperature
   3. Dilution Buffer
      1x PBS with 0.05% Tween-20, 0.1% BSA, and 0.5% methyl α-d-mannopyronoside. Keep solution at 4 °C
   4. Primary antibody solution
      Add 1:500 of rabbit anti-Streptococcus group D antigen antisera into dilution buffer. Prepare fresh every time
   5. Secondary antibody solution
      Add 1:10,000 of goat anti-rabbit IRDye 680LT into dilution buffer. Prepare fresh every time
   6. 0.5% Crystal violet solution
      Add 0.5 mg into 100 ml of distilled water. Filter sterilize the solution. Keep solution at room temperature
   7. 33% of Acetic Acid
      Add 330 ml of Acetic acid into 670 ml of distilled water. Keep solution at room temperature

Acknowledgments

C.C.W. was supported by American Heart Association GSA Predoctoral Fellowship 16PRE29860000, J.A.L. was supported by National Institute of Allergy and Infectious Disease AI135158, A.L.F.M. was supported by startup funds from the University of Notre Dame.
Special acknowledgment to previous studies done by Nallapareddy et al. (2011) and Flores-Mireles et al. (2014).

Competing interests

The authors declare no competing financial interests.

References

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