4 Notes
1. Biofilm formation has been observed in several strains including pathogenic, intermediate, and saprophyte strains. The
timeline of biofilm formation, the amount, as well as its morphology might differ according to the strain used. We recommend to calibrate the kinetic of biofilm production with regard
to the strain of interest.
2. Several types of polystyrene microtiter plates are available.
U-bottom plates or V-bottom plates are often used. We prefer
to use flat-bottom plates since they are more suitable for taking
images of the growing biofilm.
3. Petroff-Hausser cell-counting chamber 0.02 mm deep in combination with a 1.5 mm optically planed is recommended when
counting Leptospira cells. The shallow thickness of the area to
be counted limits the number of cells out of focus, improving
the counting, while the #1.5H coverslip allows a good visualization by dark-field microscopy.
Raw image
of the well
Crop and
resize
Binarization
Particle Analysis
L. interrogans
Manilae
L. biflexa
Patoc
Control
Time course (weeks)
% Area occupied by bacterial biofilm
1
2
3
10
20
30
40
50
60
0
Biofilm quantification
1
2
3
4
Fig. 3 Workflow of image analysis performed from unstained, unfixed biofilm images (1). The raw image is
cropped to removed artifacts (2), and a binary image is created (3) prior to biofilm particle quantification (4).
The graphic represents an example of quantitative biofilm data that may be obtained using this particular
image quantification method
212
Roman Thibeaux et al.
1. Biofilm formation has been observed in several strains including pathogenic, intermediate, and saprophyte strains. The
timeline of biofilm formation, the amount, as well as its morphology might differ according to the strain used. We recommend to calibrate the kinetic of biofilm production with regard
to the strain of interest.
2. Several types of polystyrene microtiter plates are available.
U-bottom plates or V-bottom plates are often used. We prefer
to use flat-bottom plates since they are more suitable for taking
images of the growing biofilm.
3. Petroff-Hausser cell-counting chamber 0.02 mm deep in combination with a 1.5 mm optically planed is recommended when
counting Leptospira cells. The shallow thickness of the area to
be counted limits the number of cells out of focus, improving
the counting, while the #1.5H coverslip allows a good visualization by dark-field microscopy.
Raw image
of the well
Crop and
resize
Binarization
Particle Analysis
L. interrogans
Manilae
L. biflexa
Patoc
Control
Time course (weeks)
% Area occupied by bacterial biofilm
1
2
3
10
20
30
40
50
60
0
Biofilm quantification
1
2
3
4
Fig. 3 Workflow of image analysis performed from unstained, unfixed biofilm images (1). The raw image is
cropped to removed artifacts (2), and a binary image is created (3) prior to biofilm particle quantification (4).
The graphic represents an example of quantitative biofilm data that may be obtained using this particular
image quantification method
212
Roman Thibeaux et al.