4. When working with crystal violet, wear gloves and a lab coat,
and be careful to avoid spilling the powder, which will readily
stain clothes and surfaces. Stains can be removed to some
degree with 70 or 95% ethanol, but it is difficult to remove all
crystal violet once it has stained a surface. It is best to dedicate a
bottle and stir bar exclusively to preparation of crystal violet
solution. Crystal violet is classified as a hazardous substance
(H226, flammable liquid and vapor; H319, causes serious eye
irritation; H351, suspected of causing cancer; H411, toxic to
aquatic life with long-lasting effects). The use of a chemical
hood while working with crystal violet is strongly advised.
5. Leptospira‘doubling time can vary from 6 to 24 h. We recommend to precisely determine the growth kinetic of the strain of
interest in order to identify the exponential growth phase. With
our strain L. interrogans Manilae L495, the exponential growth
phase is observed between the second and fifth days.
6. We recommend performing the counting from a 1/100 dilution of the initial culture. It is also useful to wait a few minutes
before starting counting the bacteria to ensure that the liquid
flow within the counting chamber is equilibrated and that no
movement of liquid will disturb the counting.
7. Border effects are very common when working with a 96-well
plate, especially if the incubator does not have a humidity
control system. Therefore we recommend to add 200 μL of
sterile distilled water in the peripheral wells of the plate.
8. Placing the covered plate in a disposable plastic storage container can reduce evaporation of media in the incubation
chamber.
9. We observed that Leptospira biofilm is weakly adherent and can
rapidly be lost when pipetting. Therefore we recommend to let
the biofilm grow under strict static conditions for at least
3 weeks when it eventually starts to be adherent (although
still fragile). Limiting the numbers of pipetting event is crucial
to avoid the loss of biofilm which might give erroneous quantitative results.
10. If there is no visible biofilm formation in any of your samples,
there are several things you may need to adjust (the length of
incubation) or check (assay medium evaporation). We also
recommend to take extra care during the pipetting steps as
they are mainly responsible for biofilm detachment.
11. During long-term incubation, condensation may form on the
lid and can impede well observation or be responsible for
artifact if pictures are taken. We recommend drying the lid
under a sterile hood for 5 min prior to sample examination.
Leptospira ´ Biofilm Quantification
213
and be careful to avoid spilling the powder, which will readily
stain clothes and surfaces. Stains can be removed to some
degree with 70 or 95% ethanol, but it is difficult to remove all
crystal violet once it has stained a surface. It is best to dedicate a
bottle and stir bar exclusively to preparation of crystal violet
solution. Crystal violet is classified as a hazardous substance
(H226, flammable liquid and vapor; H319, causes serious eye
irritation; H351, suspected of causing cancer; H411, toxic to
aquatic life with long-lasting effects). The use of a chemical
hood while working with crystal violet is strongly advised.
5. Leptospira‘doubling time can vary from 6 to 24 h. We recommend to precisely determine the growth kinetic of the strain of
interest in order to identify the exponential growth phase. With
our strain L. interrogans Manilae L495, the exponential growth
phase is observed between the second and fifth days.
6. We recommend performing the counting from a 1/100 dilution of the initial culture. It is also useful to wait a few minutes
before starting counting the bacteria to ensure that the liquid
flow within the counting chamber is equilibrated and that no
movement of liquid will disturb the counting.
7. Border effects are very common when working with a 96-well
plate, especially if the incubator does not have a humidity
control system. Therefore we recommend to add 200 μL of
sterile distilled water in the peripheral wells of the plate.
8. Placing the covered plate in a disposable plastic storage container can reduce evaporation of media in the incubation
chamber.
9. We observed that Leptospira biofilm is weakly adherent and can
rapidly be lost when pipetting. Therefore we recommend to let
the biofilm grow under strict static conditions for at least
3 weeks when it eventually starts to be adherent (although
still fragile). Limiting the numbers of pipetting event is crucial
to avoid the loss of biofilm which might give erroneous quantitative results.
10. If there is no visible biofilm formation in any of your samples,
there are several things you may need to adjust (the length of
incubation) or check (assay medium evaporation). We also
recommend to take extra care during the pipetting steps as
they are mainly responsible for biofilm detachment.
11. During long-term incubation, condensation may form on the
lid and can impede well observation or be responsible for
artifact if pictures are taken. We recommend drying the lid
under a sterile hood for 5 min prior to sample examination.
Leptospira ´ Biofilm Quantification
213