• Bacterial plating medium (blood agar). Blood agar is prepared according to manufacturer’s instructions. Plates can be stored bottom up at 4°C for maximum 2 months.
• Pre-culture media (10 ml of TSB, sterilized). TSB is prepared according to manufacturer’s instructions. Can be stored at room temperature for maximally 2 months.
• Main culture media (200 ml of TSB, sterilized). TSB is prepared according to manufacturer’s instructions. Can be stored at room temperature for maximally 2 months.
• Continuous flow media (1 l TSB, 1 l LB sterilized). TSB and LB are prepared according to manufacturer’s instructions. Can be stored at room temperature for maximally 2 months.
• Continuous flow media (1 l artificial sputum medium (ASM), Table 1). Add all components mentioned in Table 1 to 1 l of sterile demineralized water. Opposite to the other media, ASM is used immediately after preparation.
CRITICAL STEP To prevent sedimentation of ASM medium components add a sterile magnet to the medium and stir during the entire experiment.
Bacterial culture and inoculum preparation (estimated time 42 h of which 3 h active labor)
1 Obtain bacteria from a stock (in this protocol, bacteria were obtained after thawing of a frozen stock -80°C)), streak on blood agar and incubate for 24 h at 37°C to obtain single colonies.
CAUTION If using human pathogens; all experiments need to be performed within a biosafety level 2 laboratory. Use appropriate protection and decontaminate (autoclave) equipment and waste before disposal.
2 Transfer one single colony with inoculation loop and place into 10 ml of TSB, vortex, and incubate the pre-culture at 37°C for 24 h.
3 Vortex and transfer the 10 ml to 200 ml TSB (main-culture) and place at 37°C under shaking (150 RPM) for 16 h.
4 Harvest the bacteria from the main culture by centrifugation at 5,000 g for 5 min at 10°C.
5 Wash the bacteria two times by resuspending the pellet into 10 ml of PBS and centrifuge two times at 5,000 g for 5 min at 10°C.
6 Resuspend the pellet in 10 ml PBS,
7 Count the bacterial density using a Bürker-Türk counting chamber; dilute the stock if necessary for ease of counting.
8 Add the desired number of bacteria (5 x 107 bacteria/ml, 200 ml) of TSB and use as CDFF inoculum.
Prepare CDFF components (lid, scraper blade, turn table, pans, plugs, disks and side screws) for autoclaving (estimated time 16 h of which 2 h active labor)
1 Place the turn table components in a 0.2% peracetic acid bath overnight.
2 Next day take the turn table components out of the peracetic acid and rinse with demineralized water.
3 Sonicate the turn table components in 2% RBS three times 5 min in a sonicator bath, and rinse afterwards with demineralized water.
4 Place the turn table components in methanol for 5 min, and air-dry afterwards.
CAUTION Methanol is toxic; perform this in a fume hood and wear appropriate protection.
5 Wear gloves from now on to keep all surfaces clean. Place the disks on top of the plugs into the pans.
6 Set the desired well-depth of the disks using the auxiliary tool.
7 Place the pans into the turn table.
8 Fill an Erlenmeyer with 1 l of the desired growth medium. If using ASM, aseptically combine the ingredients and add to a sterile Erlenmeyer.
9 Assemble the CDFF, including silicone rubber tubing, and place it in the autoclave.
Sterilize CDFF components (estimated time 4 h)
• Autoclave the assembled CDFF and medium for 20 min at 121°C.
Settings of the CDFF (estimated time 5 min)
• Set the pump rate at the desired speed before the experiment.
• Set the pressure of the scraper.
• Set the turn table RPM before the experiment.
• Set the water bath to the desired temperature (37℃) 1 h before inoculation.
Inoculation (estimated time 1.5 h of which 20 min active labor)
1 Take the CDFF parts aseptically out of the autoclave and set up the CDFF for inoculation.
CAUTION Make sure that sample disks have not moved upwards in the wells during autoclaving (Figure 3); if so push them back to the right position with a sterile cotton bud (for example, this may happen with disks made of porous material e.g. hydroxyapatite).
2 Attach the effluent port with the silicone rubber tube to the 2 l waste Erlenmeyer filled and seal with cotton wool.
3 Set the turn table speed to 3 RPM.
4 Push the scraper to the desired applied force (40 kPa). The force blade applied was determined using a weighting scale and measuring the compression of the spring.
5 Mount the silicone rubber tubing on the pump, attach one end to the CDFF inoculation port and place the other into the inoculation broth (5 x 107 bacteria/ml, 200 ml, see reagent setup).
6 Turn on the pump at a flow rate of 200 ml/h for 1 h.
7 After 1 h, stop the pump and turn table for 30 min to allow bacterial adhesion.
Biofilm growth (estimated time 10 min plus, in our particular case 24 h for biofilm growth)
8 Attach the silicone rubber tubing from the growth medium to the CDFF.
9 Set the turn table speed at 3 RPM.
10 Switch on the pump at a flow rate of 30 ml/h and let the growth medium drip on the turn table.
11 Operate the CDFF in continuous flow for 24 h.
CAUTION Place the CDFF in a tub or tray in case of unexpected leakage.
Biofilms grown in the CDFF can be visualized using different microscopic techniques, amongst which confocal laser scanning microscopy (CLSM) is most frequently used. CLSM and other fluorescent microscopic techniques allow the use of specific fluorophores to demonstrate bacterial presence and prevalence of specific bacterial strains, possible membrane damage (viability) after antibiotic treatment, presence of eDNA and other EPS components in CDFF grown biofilms12,13 and last but not least, verify their thickness. However, fluorophores do not necessarily penetrate through the entire thickness of a biofilm, which limits the use of fluorescent techniques. Also laser light does not necessarily penetrate through an entire biofilm, which can be improved by using 2-photon laser scanning microscopy14.
CLSM after application of appropriate fluorophores, in combination with the software program COMSTAT15 is a common method to measure biofilm thickness, but a single image usually covers only a small area (0.00023 - 0.0056 cm2). Because of the limited penetration of fluorophores and laser light, CLSM will underestimate biofilm thickness for thicker biofilms as compared with other methods. Moreover, the small areas covered per image make it impossible to verify constant thickness over the entire area of a biofilm grown. Low-load compression testing (LLCT) is another technique with which biofilm thickness can be determined16 and over a larger area than with CLSM (0.049-0.57 cm2). Like CLSM, LLCT irreversibly changes the biofilm rendering them useless for further analysis. Recently, optical coherence tomography (OCT) has been introduced to visualize biofilms in their hydrated state, non-destructively and in real-time over a large area (up to several cm2), albeit with limited resolution17. 3D images obtained from the OCT can be analyzed using a custom LabVIEW script, allowing calculation of the average biofilm thickness. In Figure 4 we present a comparison of biofilm thicknesses obtained using different methods.
OCT Biofilm visualization and determination of biofilm thickness and biofilm height distribution (estimated time 30 min per pan, i.e. 5 biofilms)
12 Stop the turn table and pump, open the sample port and take one pan containing the sample disks with biofilm aseptically out of the CDFF with the help of the sampling tool. Close the sampling port until further use and continue the experiment.
13 Carefully immerse the pan, or screw the disks out of the pans, and place in a sample container filled with PBS.
CRITICAL STEP Handle the pan and disks gently because the biofilms may detach if disturbed vigorously when the biofilm is placed in PBS.
14 Start up the OCT and the Thor Image software.
15 Make sure the right refractive index is used and the physical depth is high enough to visualize the complete biofilm. For 3D images use field of view (FOV) of 6,000 µm x 6,000 µm x 1,000 µm, with a size of 1,500 x 300 x 373 (pixels). For 2D images use FOV 6,000 µm x 1,000 µm, with a size of 5,000 x 373 (pixels).
16 Take 2D or/and 3D images of the biofilms.
CRITICAL STEP The OCT imaging system should stand on a stable, vibration free table, since the measurements are very sensitive to mechanical disturbances.
17 After taking the images, biofilms can be used for further analysis.
18 3D images obtained from the OCT can be analyzed using a custom LabVIEW script. This script allows the precise calculation of the average biofilm thickness over a specific region of interest (ROI). Import the 3D OCT file into the LabVIEW script. A single 3D OCT image contains a certain number of 2D OCT images, this number depends on the chosen settings while obtaining the OCT image (in our case the number was 300).
19 Select a square ROI from the 3D OCT image.
20 Indicate the substratum in the image using the fact that the substratum has the highest pixel intensity (in our case because we use stainless steel as substratum).
CRITICAL STEP Pixels with highest intensity may not be the indicator of the substratum because not every substratum is as reflective as metals (in our case stainless steel). For non-metallic substrata, the substratum has to be more carefully indicated based on another suitable criterion.
21 Divide each 2D image into vertical slices by selecting the slice width; we take a slice every 20 pixels.
22 Carefully check if the automatic threshold chosen by the program correctly indicates the top of the biofilm. The program automatically selects a grey scale threshold to differentiate between the biofilm top and the overlying fluid. The 5% of the pixels with the highest intensity are selected (white pixels, biofilm), and the 5% of the pixels with the lowest intensity are selected (black pixels, background). With this information, a threshold is set. The threshold can be adjusted manually if necessary.
23 Start the calculation of the parameters defining the biofilm thickness and biofilm height distribution over the entire surface area of a biofilm (Figure 5).
24 When the experiments are finished, autoclave the complete CDFF with the components and the biofilms 15 min at 134°C to sterilize the CDFF.