Materials and Reagents

0.2 μm filter
Bacteriological Petri plates (Fisher Scientific, catalog number: FB0875712 )
Sterile test tubes (17 x 100 mm, RPI, Research Products International, catalog number: 168599 )
Serological pipettes:
5 ml pipette (SARSTEDT, catalog number: 86.1253.001 )
25 ml pipette (SARSTEDT, catalog number: 86.1685.001 )
Nalgene^TM 2 ml cryogenic tubes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number 5000-0020 )
Coverglasses, 22 x 22 mm (Fisher Scientific, Fisherbrand^TM, catalog number: 12-542B )
Nunc^TM 6-wells plates (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 150239 )
Aluminum foil
Microscope slides (Fisher Scientific, Fisherbrand^TM, catalog number: 12-552-3 )
Sterile disposable plastic material:
Posi-Click tubes (Denville Scientific, catalog number: C2170 )
P200 and P1000 tips (USA Scientific, catalog numbers: 1111-0706 and 1112-1720 )
B. pertussis strain harboring a pGB5P1-GFP plasmid
Defibrinated sheep’s blood (Hemostat Laboratories, catalog number: DSB100 )
50% glycerol solution (Fisher Scientific, catalog number: G33-1 )
Kanamycin (Sigma-Aldrich, catalog number: K1377 )
Neutralized buffered formalin (Fisher Scientific, catalog number: SF100-4 )
Stainer-Scholte medium (Stainer et al., 1970; Nicholson et al., 2012)
Sterile PBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190144 )
ProLong^TM Gold Antifade Mountant (Thermo Fisher Scientific, Invitrogen^TM, catalog number: P36934 )
Difco^TM Bordet-Gengou agar plates (BD, BD Biosciences, catalog number: 248200 )
L-Proline (Sigma-Aldrich, catalog number: 131547 )
KH₂PO₄ (Sigma-Aldrich, catalog number: V000225 )
KCl (Sigma-Aldrich, catalog number: P3911 )
MgCl₂·6H₂O (Sigma-Aldrich, catalog number: M2393 )
CaCl₂ (Sigma-Aldrich, catalog number: C1016 )
Tris base (Sigma-Aldrich, catalog number: T1378 )
NaCl (Sigma-Aldrich, catalog number: S7653 )
FeSO₄·7H₂O (Sigma-Aldrich, catalog number: F8633 )
L-cystine (Sigma-Aldrich, catalog number: C8755 )
L-ascorbic acid (Sigma-Aldrich, catalog number: A5960 )
Nicotinic acid (Sigma-Aldrich, catalog number: PHR1276 )
Reduced L-glutathione (Sigma-Aldrich, catalog number: G4251 )
SS medium (see Recipes)
SS supplement (filter-sterilized) (see Recipes)
Bordet-Gengou (BG) medium (see Recipes)

Equipment

Kimax^® Erlenmeyer flasks (125 ml) (DWK Life Sciences, KIMBLE^®, catalog number: 26500 )
Sterile forceps
PIPETMAN^® Classic (Gilson, models: P20, P200, P1000, catalog numbers: F123600 , F123601 , F123602 )
Humid chamber (an appropriately sized Tupperware container with either a weigh boat containing water or paper towels soaked with water)
Chemical fume hood (Hamilton Laboratory Solutions, model: PL-822 )
Eclipse Ti-E inverted Confocal Microscope (Nikon, model: D-Eclipse C1si )
Vortex (Fisher Scientific, catalog number: 02-215-365 )
pH meter (Denver Instrument, model: UB-10 )
Incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Forma^TM Steri-Cycle^TM )
Benchtop centrifuge (Eppendorf, model: 5418 )
Refrigerator (4 °C) (Whirlpool, model: WRR56X18FW02 )
-20 °C freezer (Kenmore, model: 253-26722103 )
SPECTRONIC ^TM Spectrophotometer (Thermo Fisher Scientific, model: GENESYS 20 )
Weigh Balance (Ohaus, model: Scout Pro SP202 and VWR, model: VWR-164AC )
Roller Drum (Eppendorf, New Brunswick^TM, model: TC-2 )

Software

ImageJ (Schneider et al., 2012)
COMSTAT2 plugin (Heydorn et al., 2000)

Procedure

Notes:

The entire protocol is depicted in Figure 1.
Samples should be protected from light during the experiment. In all cases, medium is supplemented with appropriate antibiotic to maintain the plasmid coding for GFP.

Figure 1. Schematic step-by-step protocol for growing B. pertussis biofilms for CLSM evaluation

Growth of B. pertussis strains
1. From frozen stock stored at -78 °C, streak B. pertussis strains harboring a GFP-coding plasmid, pGB5P1-GFP was used in this protocol (Weingart et al., 1999), on Bordet-Gengou agar plates supplemented with 10% defibrinated sheep’s blood and 25 μg/ml of kanamycin. Incubate the plates at 37 °C for 4-5 days.
2. Pick individual colonies (~8-10) and inoculate 2 ml of Stainer-Scholte (SS) liquid medium supplemented with 25 μg/ml of kanamycin. Incubate at 37°C in a roller drum spinning at top speed for 24 h.
3. Inoculate 30 ml of SS medium supplemented with 25 μg/ml of kanamycin (in a 100 ml Erlenmeyer flask), with the cultures from Step A2 to achieve an OD₆₅₀ of 0.2. Incubate for 12 h at 37 °C with shaking at 160 rcf.
In vitro biofilm formation
1. Insert a sterile coverglass (flame sterilized) at the bottom of sterile 6-well plates.
2. Prepare inoculum by diluting B. pertussis culture (Procedure A, Step 3) to an OD₆₅₀ of 1.0 in SS medium. Pipet 1.5 ml of inoculum into individual wells of the 6-well plates. Make sure the bacterial suspension is covering the entire surface of the coverslip. In case other substrates are used (i.e., polycarbonate or aluminum coupons), volume should be adjusted according to the thickness of the material.
3. Cover the plate and incubate statically in a humidified chamber for 4 h at 37 °C.
  Note: This step allows the adhesion of bacteria to the surface.
4. Remove inocula (to allow removal of unattached planktonic bacteria) carefully without disturbing attached cells and add approximately 800 μl of fresh SS medium, dispensing along the wall of the well to prevent biofilm disruption. Place the plate at an angle of 30°-50° relative to horizontal, forming an air-liquid interface in the coverglass where biofilms will develop. Cover the plate with aluminum foil and incubate in a humidified chamber at 37 °C on a platform shaker at 90 rcf.
5. If the biofilm formation is going to be examined for longer than 24 h, change medium every 24 h as described above. Samples can be processed at 24, 48, and 72 up to 96 h.
Visualization of biofilms
1. After each time point, aspirate medium from each well and carefully wash the biofilms twice with sterile PBS.
2. Pick the coverglass up with forceps and place it into a new 6-well plate. A needle can be used to aid in lifting the coverglass followed by use of forceps to pick the coverglass without touching the area where the biofilm developed.
3. Carefully add 50 μl of neutral buffered formalin, or enough to cover the glass surface. Special attention must be taken in order to not disturb the biofilm and incubate for 15 min at room temperature.
4. Remove fixative and wash twice with PBS.
5. Pick the coverglass up with forceps and mount onto a glass slide with ProLong^TM Gold mounting media. Alternatively, if bacteria are not labeled with a fluorescent protein, cells can be stained at this point with a fluorescent dye and then mounted.
6. Allow the mounting media to harden by incubating at 4 °C for 24 h.
7. Slides can be stored (weeks to months) in the dark at room temperature for later visualization. Examine the samples with a confocal microscope, taking images in a z-stack configuration, making sure to cover the entire biofilm structure from bottom to top.
8. Analyze the images by using microscopy image visualization software (Figure 2).
  
  Figure 2. Micrographs of a B. pertussis biofilm. Biofilms were grown from 24 up to 96 h and images were acquired in an inverted confocal microscope in a z-stack setting. Images are presented in xy, xz and yz planes.

Data analysis

Quantitative data from images can be obtained with COMSTAT2 plug-in (Heydorn et al., 2000) run in ImageJ (Schneider et al., 2012). Structural features that can be analyzed are maximum and average thicknesses, substrate coverage, biomass and roughness coefficient (see structural data in Table 1). Detailed instructions for COMSTAT2 use are described in Heydorn et al. (2000). Appropriate statistical analysis should be carried out on the obtained data.

Table 1. Biofilm structural features of the images presented in Figure 2, obtained with COMSTAT2 software

Notes

Due to variations observed with this assay, there is need to be performed with duplicates of each sample and at least three independent replicates. For each sample, at least three representative images should be acquired.
A critical consideration is not to disrupt the biofilm attached to the glass surface. Depending on the microorganism, cells may attach either strongly or loosely to surfaces. Thus it is important to be extremely careful while dispensing liquid to avoid strong shear forces.
BG-agar medium and SS medium are sterilized by autoclaving at 121 °C for 15 min.
To prepare a cryogenic stock, cultures grown to exponential phase (set up as explained in Step A2) should be used. Use sterile 2 ml cryogenic tubes and mix 500 μl of bacterial culture with 500 μl of sterile 50% glycerol solution. Store at -78 °C.

Recipes

Note: Unless otherwise indicated, all stock solutions are prepared using Milli Q water.

SS medium
Monosodium glutamate: 10.70 g/L
L-Proline: 0.24 g/L
KH₂PO₄: 0.5 g/L
KCl: 0.20 g/L
MgCl₂·6H₂O: 0.20 g/L
CaCl₂: 0.2 g/L
Tris base: 1.52 g/L
NaCl: 2.50 g/L
Adjust to pH 7.3-7.4 with HCl
SS supplement (0.2 μm filter-sterilized)
Dissolve in HCl concentrated, and then filter sterilize
FeSO₄·7H₂O: 10 mg/L
L-cystine: 40 mg/L
L-ascorbic acid: 20 mg/L
Nicotinic acid: 4 mg/L
Reduced L-glutathione: 100 mg/L
Bordet-Gengou (BG) medium
Difco^TM Bordet-Gengou agar (30 g/L)
20 ml of 50% glycerol
Autoclave at 121 °C for 15 min
Aseptically add 15% sterile, defibrinated blood to the medium at 45-50 °C
Mix well and plate

Acknowledgments

This project has been funded in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services by grants 1R21AI123805-0 and R01AI125560.
This protocol was adapted from Cattelan, N., Jennings-Gee, J., Dubey, P., Yantorno, O. M. and Deora, R. (2017). Hyperbiofilm Formation by Bordetella pertussis Strains Correlates with Enhanced Virulence Traits. Infect Immun 85(12).
The authors declare no conflict of interest.

References

Cattelan, N., Dubey, P., Arnal, L., Yantorno, O. M. and Deora, R. (2016). Bordetella biofilms: a lifestyle leading to persistent infections. Pathog Dis 74(1): ftv108.
Cattelan, N., Jennings-Gee, J., Dubey, P., Yantorno, O. M. and Deora, R. (2017). Hyperbiofilm formation by Bordetella pertussis strains correlates with enhanced virulence traits. Infect Immun 85(12).
Conover, M. S., Mishra, M. and Deora, R. (2011). Extracellular DNA is essential for maintaining Bordetella biofilm integrity on abiotic surfaces and in the upper respiratory tract of mice. PLoS One 6(2): e16861.
Conover, M. S., Sloan, G. P., Love, C. F., Sukumar, N. and Deora, R. (2010). The Bps polysaccharide of Bordetella pertussis promotes colonization and biofilm formation in the nose by functioning as an adhesin. Mol Microbiol 77(6): 1439-1455.
Dorji, D., Mooi, F., Yantorno, O., Deora, R., Graham, R. M. and Mukkur, T. K. (2018). Bordetella pertussis virulence factors in the continuing evolution of whooping cough vaccines for improved performance. Med Microbiol Immunol 207(1): 3-26.
Ganguly, T., Johnson, J. B., Kock, N. D., Parks, G. D. and Deora, R. (2014). The Bordetella pertussis Bps polysaccharide enhances lung colonization by conferring protection from complement-mediated killing. Cell Microbiol 16(7): 1105-1118.
Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K. and Molin, S. (2000). Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146 (Pt 10): 2395-2407.
Mishra, M., Parise, G., Jackson, K. D., Wozniak, D. J. and Deora, R. (2005). The BvgAS signal transduction system regulates biofilm development in Bordetella. J Bacteriol 187(4): 1474-1484.
Mooi, F. R. (2010). Bordetella pertussis and vaccination: the persistence of a genetically monomorphic pathogen. Infect Genet Evol 10(1): 36-49.
Nicholson, T. L., Conover, M. S. and Deora, R. (2012). Transcriptome profiling reveals stage-specific production and requirement of flagella during biofilm development in Bordetella bronchiseptica. PLoS One 7(11): e49166.
Parise, G., Mishra, M., Itoh, Y., Romeo, T. and Deora, R. (2007). Role of a putative polysaccharide locus in Bordetella biofilm development. J Bacteriol 189(3): 750-760.
Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
Serra, D. O., Conover, M. S., Arnal, L., Sloan, G. P., Rodriguez, M. E., Yantorno, O. M. and Deora, R. (2011). FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea. PLoS One 6(12): e28811.
Serra, D. O., Lucking, G., Weiland, F., Schulz, S., Gorg, A., Yantorno, O. M. and Ehling-Schulz, M. (2008). Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis. Proteomics 8(23-24): 4995-5010.
Sloan, G. P., Love, C. F., Sukumar, N., Mishra, M. and Deora, R. (2007). The Bordetella Bps polysaccharide is critical for biofilm development in the mouse respiratory tract. J Bacteriol 189(22): 8270-8276.
Stainer, D. W. and Scholte, M. J. (1970). A simple chemically defined medium for the production of phase I Bordetella pertussis. J Gen Microbiol 63(2): 211-220.
Sukumar, N., Love, C. F., Conover, M. S., Kock, N. D., Dubey, P. and Deora, R. (2009). Active and passive immunizations with Bordetella colonization factor A protect mice against respiratory challenge with Bordetella bronchiseptica. Infect Immun 77(2): 885-895.
Weingart, C. L., Broitman-Maduro, G., Dean, G., Newman, S., Peppler, M. and Weiss, A. A. (1999). Fluorescent labels influence phagocytosis of Bordetella pertussis by human neutrophils. Infect Immun 67(8): 4264-4267.