8. At this point, you should be able to see dot-like or reticulated
structures at the bottom of the wells (see Note 10). The wells
can be photographed for qualitative illustration (see Fig. 2a).
3.3 CV-Stained
Biofilm Quantitation
1. Pipette 200 μL of 30% acetic acid solution into each well. This
will solubilize the CV.
2. Incubate for 10–15 min.
3. Pipette up and down to assure that the stained biofilm is well
solubilized, and then transfer 100 μL of each sample to a new
96-well optically clear, flat-bottom plate.
4. Read optical density of all samples in plate at a wavelength of
550–600 nm [15].
5. Plot and analyze the data (example given in Fig. 2b).
3.4 Biofilm Phase
Contrast Image
Quantitation
Alternative to CV quantitation, we also found useful to directly
quantify the biofilm formation over time through phase contrast
image analysis. This method significantly reduces sample manipulation and allows to work with native, unfixed, unstained biofilm.
1. Every week, take phase contrast images of the growing biofilm
(see Note 11). Make sure that the center of the well is into
focus. Typically, we take pictures of six replicate wells per
independent experiment.
2. Using an image analysis software, crop the outer part of the
image where spherical aberrations and field curvature artifacts
are visible (see Note 12).
3. Create a binary image from your 2D grayscale image and using
the particle analysis tool quantify the surface of the biofilm as a
function of time (see Fig. 3).
Time course (weeks)
Absorbance (O.D.)
1
2
3
0.2
0.4
0.6
Leptospira interrogans Manilae
30 % acetique acide
Leptospira biflexa Patoc
0
Microtiter Plate Biofilm Assay
A
B
Fig. 2 (a) Phase contrast images illustrating crystal violet staining of Leptospira biofilm after 3 weeks of
incubation. (b) Example of data that may be obtained by using spectrophotometry to quantitate biofilm
formation
Leptospira ´ Biofilm Quantification
211
structures at the bottom of the wells (see Note 10). The wells
can be photographed for qualitative illustration (see Fig. 2a).
3.3 CV-Stained
Biofilm Quantitation
1. Pipette 200 μL of 30% acetic acid solution into each well. This
will solubilize the CV.
2. Incubate for 10–15 min.
3. Pipette up and down to assure that the stained biofilm is well
solubilized, and then transfer 100 μL of each sample to a new
96-well optically clear, flat-bottom plate.
4. Read optical density of all samples in plate at a wavelength of
550–600 nm [15].
5. Plot and analyze the data (example given in Fig. 2b).
3.4 Biofilm Phase
Contrast Image
Quantitation
Alternative to CV quantitation, we also found useful to directly
quantify the biofilm formation over time through phase contrast
image analysis. This method significantly reduces sample manipulation and allows to work with native, unfixed, unstained biofilm.
1. Every week, take phase contrast images of the growing biofilm
(see Note 11). Make sure that the center of the well is into
focus. Typically, we take pictures of six replicate wells per
independent experiment.
2. Using an image analysis software, crop the outer part of the
image where spherical aberrations and field curvature artifacts
are visible (see Note 12).
3. Create a binary image from your 2D grayscale image and using
the particle analysis tool quantify the surface of the biofilm as a
function of time (see Fig. 3).
Time course (weeks)
Absorbance (O.D.)
1
2
3
0.2
0.4
0.6
Leptospira interrogans Manilae
30 % acetique acide
Leptospira biflexa Patoc
0
Microtiter Plate Biofilm Assay
A
B
Fig. 2 (a) Phase contrast images illustrating crystal violet staining of Leptospira biofilm after 3 weeks of
incubation. (b) Example of data that may be obtained by using spectrophotometry to quantitate biofilm
formation
Leptospira ´ Biofilm Quantification
211