samples, the ease of replication, and the variety of samples that can
be tested in a single assay.
In the classical procedure, bacterial cells are grown in the wells
of a polystyrene microtiter plate [6].
At different time points, the wells are washed to remove planktonic cells before staining the biofilm attached to the surface of the
wells. The most widely used method to follow biofilm formation in
microtiter plates is the crystal violet staining method [7], which
measures biofilm biomass at the bottom of the well. Crystal violet
(CV) belongs to the family of triphenylmethane dyes, which bind to
the bacterial cellular components by ionic interactions. CV is a basic
dye that stains both live and dead cells by binding to negatively
charged surface molecules including DNA [8], proteins [9], and
polysaccharides [10]. Quantitation of biofilm growth can be
obtained by solubilization of the CV and measurement of the
absorbance using a spectrophotometer. This approach has been
successfully used to characterize in vitro the initial step of Leptospira
biofilm formation [11].
In this chapter, we also detail an alternative method based on
phase contrast image quantification instead of CV quantification.
We found this method as robust as the CV method to quantify
biofilm formation, but most importantly, the latter has the advantage of limiting sample handling and thereby reducing erroneous
quantification resulting from biofilm disruption during washing
steps. Nevertheless, the need for a good microscope and a CCD
camera may appear to be an obstacle for some laboratories.
2 Materials
2.1 Biofilm Growing
1. Actively motile exponential-phase Leptospira interrogans serovar Manilae strain L495 (see Note 1).
2. Ellinghausen-McCullough-Johnson-Harris (EMJH) culture
medium [12, 13]: Dissolve 2.3 g of Difco™ Leptospira
Medium Base EMJH (Becton Dickinson) in 900 mL of purified water. Add 100 mL of Difco™ Leptospira Enrichment
EMJH (Becton Dickinson) (see Chapter 1).
3. 96-well optically clear, sterile polystyrene flat-bottom plate
with low evaporation lid (see Note 2).
4. Flat-bottom, sterile glass culture tubes with screw cap.
5. 5 mL Combitips advanced.
6. Multipette plus.
7. Petroff-Hausser cell-counting chamber (see Note 3).
8. Upright dark-field microscope with a 20Â objective.
9. 30
C incubator with controlled humidity (see Notes 7 and 8).
10. Lab coat and nitrile gloves.
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Roman Thibeaux et al.
be tested in a single assay.
In the classical procedure, bacterial cells are grown in the wells
of a polystyrene microtiter plate [6].
At different time points, the wells are washed to remove planktonic cells before staining the biofilm attached to the surface of the
wells. The most widely used method to follow biofilm formation in
microtiter plates is the crystal violet staining method [7], which
measures biofilm biomass at the bottom of the well. Crystal violet
(CV) belongs to the family of triphenylmethane dyes, which bind to
the bacterial cellular components by ionic interactions. CV is a basic
dye that stains both live and dead cells by binding to negatively
charged surface molecules including DNA [8], proteins [9], and
polysaccharides [10]. Quantitation of biofilm growth can be
obtained by solubilization of the CV and measurement of the
absorbance using a spectrophotometer. This approach has been
successfully used to characterize in vitro the initial step of Leptospira
biofilm formation [11].
In this chapter, we also detail an alternative method based on
phase contrast image quantification instead of CV quantification.
We found this method as robust as the CV method to quantify
biofilm formation, but most importantly, the latter has the advantage of limiting sample handling and thereby reducing erroneous
quantification resulting from biofilm disruption during washing
steps. Nevertheless, the need for a good microscope and a CCD
camera may appear to be an obstacle for some laboratories.
2 Materials
2.1 Biofilm Growing
1. Actively motile exponential-phase Leptospira interrogans serovar Manilae strain L495 (see Note 1).
2. Ellinghausen-McCullough-Johnson-Harris (EMJH) culture
medium [12, 13]: Dissolve 2.3 g of Difco™ Leptospira
Medium Base EMJH (Becton Dickinson) in 900 mL of purified water. Add 100 mL of Difco™ Leptospira Enrichment
EMJH (Becton Dickinson) (see Chapter 1).
3. 96-well optically clear, sterile polystyrene flat-bottom plate
with low evaporation lid (see Note 2).
4. Flat-bottom, sterile glass culture tubes with screw cap.
5. 5 mL Combitips advanced.
6. Multipette plus.
7. Petroff-Hausser cell-counting chamber (see Note 3).
8. Upright dark-field microscope with a 20Â objective.
9. 30
C incubator with controlled humidity (see Notes 7 and 8).
10. Lab coat and nitrile gloves.
208
Roman Thibeaux et al.