2.2 Biofilm
Quantification
2.2.1 Crystal Violet
Quantification (See
Note 4)
1. 0.1% Crystal Violet (CV) solution: 0.1% CV in water.
2. Phosphate-buffered saline (PBS): 10 mM Na 2 PO 4 , 1.8 mM
KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4.
3. Fixative buffer: 4% paraformaldehyde (PFA) in PBS.
4. Pipetter.
5. Multichannel pipette.
6. 200 μL tips.
7. Multichannel pipette reservoir.
8. Container for liquid waste.
9. 30% glacial acetic acid solution: 30% glacial acetic acid in water.
10. 96-well optically clear, flat-bottom plate (can be non-sterile).
11. Spectrophotometer equipped with plate reading capability
(550–600 nm).
12. Timer.
13. Deionized H 2 O.
2.2.2 Phase Contrast
Image Quantification
1. Upright phase contrast microscope with a 5Â objective and
equipped with a CCD digital camera.
2. Open-source Image Software analysis Fiji [14] (https://fiji.sc/).
3 Methods
3.1 Growing
Leptospira Biofilm
1. Inoculate bacteria into 5 mL EMJH growth medium in sterile
culture tubes and grow to exponential phase. Typically, bacteria
are grown at 30
C for 4 days without shaking (see Note 5).
2. Count bacteria under a dark-field microscope using a PetroffHausser cell-counting chamber (see Note 6), and dilute the
initial culture with fresh EMJH to a final concentration of
1 Â 10
6 bacteria per mL.
3. Under sterile conditions, aliquot 150 μL of diluted cultures
into the wells of a 96-well microtiter plate. Be sure to include
blank wells containing sterile EMJH medium as a control. It is
recommended not to use the peripheral wells of the plate, as
border effect is known to occur (see Fig. 1 and Note 7). For
quantitative assays, we typically use 6–18 replicate wells for
each treatment.
4. Cover the plate (see Note 8), and incubate at 30
C for several
weeks in static conditions without changing the medium [11]
(see Note 9). When working with pathogenic strains, an incubation of 3 weeks is encouraged to obtain an adherent, mature
biofilm.
Leptospira ´ Biofilm Quantification
209
Quantification
2.2.1 Crystal Violet
Quantification (See
Note 4)
1. 0.1% Crystal Violet (CV) solution: 0.1% CV in water.
2. Phosphate-buffered saline (PBS): 10 mM Na 2 PO 4 , 1.8 mM
KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4.
3. Fixative buffer: 4% paraformaldehyde (PFA) in PBS.
4. Pipetter.
5. Multichannel pipette.
6. 200 μL tips.
7. Multichannel pipette reservoir.
8. Container for liquid waste.
9. 30% glacial acetic acid solution: 30% glacial acetic acid in water.
10. 96-well optically clear, flat-bottom plate (can be non-sterile).
11. Spectrophotometer equipped with plate reading capability
(550–600 nm).
12. Timer.
13. Deionized H 2 O.
2.2.2 Phase Contrast
Image Quantification
1. Upright phase contrast microscope with a 5Â objective and
equipped with a CCD digital camera.
2. Open-source Image Software analysis Fiji [14] (https://fiji.sc/).
3 Methods
3.1 Growing
Leptospira Biofilm
1. Inoculate bacteria into 5 mL EMJH growth medium in sterile
culture tubes and grow to exponential phase. Typically, bacteria
are grown at 30
C for 4 days without shaking (see Note 5).
2. Count bacteria under a dark-field microscope using a PetroffHausser cell-counting chamber (see Note 6), and dilute the
initial culture with fresh EMJH to a final concentration of
1 Â 10
6 bacteria per mL.
3. Under sterile conditions, aliquot 150 μL of diluted cultures
into the wells of a 96-well microtiter plate. Be sure to include
blank wells containing sterile EMJH medium as a control. It is
recommended not to use the peripheral wells of the plate, as
border effect is known to occur (see Fig. 1 and Note 7). For
quantitative assays, we typically use 6–18 replicate wells for
each treatment.
4. Cover the plate (see Note 8), and incubate at 30
C for several
weeks in static conditions without changing the medium [11]
(see Note 9). When working with pathogenic strains, an incubation of 3 weeks is encouraged to obtain an adherent, mature
biofilm.
Leptospira ´ Biofilm Quantification
209