Journal of Microbiological Methods 68 (2007) 605 612

www.elsevier.com/locate/jmicmeth

A new device for rapid evaluation of biofilm formation potential by bacteria

Patrick Chavant a , Brigitte Gaillard-Martinie b , Rgine Talon c ,
Michel Hbraud c,, Thierry Bernardi a
a
BioFilm Control SAS, Biople Clermont-Ferrand Limagne, F-63360 St Beauzire, France
b
Institut National de la Recherche Agronomique, site de Theix, UR454 Microbiologie, Plate-Forme Microscopie Electronique,
F-63122 Saint-Gens Champanelle, France
c
Institut National de la Recherche Agronomique, site de Theix, UR454 Microbiologie, Equipe Qualit et Scurit des Aliments,
F-63122 Saint-Gens Champanelle, France
Received 27 July 2006; received in revised form 7 November 2006; accepted 20 November 2006
Available online 9 January 2007

Abstract

This work describes the implementation of a new assay named the BioFilm Ring Test to evaluate the ability of bacteria to form biofilms. This
assay is based on the immobilisation (or not) of magnetic beads embedded by bacterial aggregates or mats (pattented concept). It is realised on
modified polystyrene 96-wells microtiter plates with individual 8-wells slides. The kinetic of biofilm formation of four bacterial species, Listeria
monocytogenes, Escherichia coli, Staphylococcus carnosus and Staphylococcus xylosus was evaluated with this new device by comparison with
the standard crystal violet staining method of sessile cells. In parallel, the biofilm growth was visualized by Scanning Electron Microscopy (SEM)
observations. The BioFilm Ring Test gave similar but faster results than the crystal violet method. Moreover, the new assay was easier to
implement, more reproducible and allowed high throughput screenings due to limited manipulations (no washing and staining steps) and rapid and
accurate measurements of magnetic bead immobilisation by sessile bacterial cells.
2006 Elsevier B.V. All rights reserved.

Keywords: Bacteria; Adhesion and biofilm formation ability; Screening device; BioFilm Ring Test

1. Introduction One of these remarkable properties is the increased resistance of

Bacterial biofilms are generally described as surface-
associated bacterial community forming microcolonies sur-
rounded by a matrix of exopolymers (Costerton and Lewan-
dowski, 1995). Microbial biofilms can exist as aggregates more
or less confluent, single layer mat or three-dimensional
architecture with channels allowing liquid and gas flow and
dispersion of nutrients and waste components (Stoodley et al.,
2002). Such structures can develop on many abiotic and biotic
surfaces (Lasa et al., 2005) and constitute the main bacterial
reservoir in most ecosystems (Costerton et al., 1999). Once
established, sessile bacteria express genes in a pattern that
greatly differs from their planktonic counterparts, leading to
phenotypic changes (Prigent-Combaret and Lejeune, 1999).
Corresponding author. Tel.: +33 4 73 62 46 70; fax: +33 4 73 62 45 81.
E-mail address: hebraud@clermont.inra.fr (M. Hbraud).
0167-7012/$ - see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2006.11.010
sessile cells to host defences, biocides, antibiotics and various
physicochemical agents (Costerton, 2005). Thus, cells in
biofilms can persist and survive even after decontamination
procedures and may represent the original source for human and
animal infections or foodstuff (re)contaminations.
Problems of public health caused by bacterial biofilms have
emphasized the lack of information about their development.
Some screening methods are available for evaluating the
ability of bacterial strains to adhere and to test their
susceptibility to antimicrobial agents. Among them are
observations by microscopy techniques (epifluorescence,
laser-scanning confocal, transmission electron and scanning
electron microscopy) or enumeration of sessile bacteria after
detachment from the surface by scraping, vortexing, sonication
or the use of beads (Zips et al., 1990; Lindsay and Von Holy,
1997; Moltz and Martin, 2005). These methods allow getting
detailed information but they are fastidious and/or time
606 P. Chavant et al. / Journal of Microbiological Methods 68 (2007) 605612
consuming and consequently not really adapted to large scale
screening experiments. One of the most convenient techniques
to evaluate bacterial adhesion consists in a colorimetric
method after washing, staining and destaining of sessile cells
with crystal violet or safranin, in tubes or microtiter plates. It
allows to screen several strains or mutants simultaneously
Genevaux et al., 1996; Vidal et al., 1998; Stepanovic et al.,
2000; Djordjevic et al., 2002; Stepanovic et al., 2004; Joseph
and Wright, 2004) or to test one microorganism under different
environmental factors (O'Toole and Kolter, 1998; Moltz and
Martin, 2005). This technique requires at least 24 to 48 h-
cultures and estimates the adhered cells stained after washing
steps. These washing steps, used to remove non-adherent cells,
are particularly critical because they can considerably
influence the final result. Moreover, some important differ-
ences from one laboratory to another exist in practice of these
washing steps, which make difficult the comparison of the
results.
Here we present a new easy to handle and rapid assay, the
BioFilm Ring Test, for the evaluation of bacterial biofilm
formation without any washing and staining steps. This device
has been validated on four bacterial strains. (i) Listeria
monocytogenes, a Gram-positive bacillus and a major food-
borne pathogen, known as a biofilm-positive strain producing
very few polymers (Wirtanen and Mattila-Sandholm, 1993),
tested on numerous surfaces such as stainless steel (Herald and
Zottola, 1988), PTFE (Chavant et al., 2002), polystyrene and
other surfaces currently used in food processing environments
(Blackman and Frank, 1996); (ii) Escherichia coli, a Gram-
negative bacillus able to form biofilms on different surfaces
such as stainless steel (Jayaraman et al., 1998), glass or
polystyrene (Prigent-Combaret and Lejeune, 1999); (iii) Sta-
phylococcus xylosus, a Gram-positive cocci used as a starter in
several food products (Talon et al., 2002), characterised as a
biofilm-positive strain on polystyrene substratum and secreting
large amounts of polysaccharides (Planchon et al., 2006); (iv)
Staphylococcus carnosus, a Gram-positive cocci used as starter
culture for sausage manufacturing and known as a biofilm-
negative strain on polystyrene surfaces (Fey et al., 1999; Gross
et al., 2001; Rupp and Fey, 2001; Gotz, 2002; Planchon et al.,
2007).
The assay, based on the immobilisation of magnetic beads
by adherent cells, was compared to the crystal violet
method and visualized by Scanning Electron Microscopy
observations.
2. Materials and methods
2.1. Bacterial strains and growth conditions
The strains studied were: L. monocytogenes EGDe, serovar
1/2 a, whose genome was recently sequenced (Glaser et al.,
2001), S. xylosus DSM 20267 isolated from human skin,
S. carnosus TM300 (provided by F. Gtz, University of
Tuebingen, Germany) and E. coli DH5.
Strains were grown in Brain Heart Infusion medium (BHI,
Difco, Detroit, MI, USA) at 150 rpm in an orbital shaking
waterbath (Aquatron, Infors, HT Switzerland), at 30 C for
the Staphylococci strains and 37 C for L. monocytogenes
and E. coli. A 25-ml preculture was incubated overnight and
used to inoculate a 25-ml culture to a final optical density at
600 nm (OD600) adjusted to 0.1 (Shimadzu UV160 A
spectrophotometer, Roucaire, Courtaboeuf, France). Cultures
were incubated until they reach the stationary phase, i.e.
about 18 h.
2.2. Preparation of Initial Bacterial Suspension (IBS)
Bacterial cells were harvested by centrifugation at 7000 g
for 10 min (MR22i, Jouan, Nantes, France) and resuspended in
sterile BHI medium to obtain an OD600 = 0.9. Then, 50 l were
inoculated in 12.1 ml of sterile BHI medium corresponding to
the Initial Bacterial Suspension (IBS) for each bacterial strain.
The same IBS was used for the BioFilm Ring Test, and for the
crystal violet assay.
2.3. Biofilm evaluation with the BioFilm Ring Test
BioFilm Control (Saint Beauzire, France) provides a kit
including: microplates (12 polystyrene strips of 8 wells, SBS
format), Toner (magnetic beads solution), Contrast Liquid (a
non-toxic and inert opaque oil used for reading step),
dedicated Block Test (magnet support) and Plate Reader
(scanner).
The Toner was added in each IBS to get a final concentration
of 6 l ml 1. This mixture was homogenized by vortexing and
200 l per well were loaded. One strip was used for each
incubation time tested. The cultures were incubated at 37 C for
L. monocytogenes EGDe and E. coli DH5 and at 30 C for
Staphylococci strains. After respectively 0, 2, 4, 6, 8, 24 and
48 h, wells of one strip were first covered with 100 l of
Contrast Liquid and scanned with the Plate Reader, a dedicated
scanner apparatus, to get an I0 image. Then, the strip was placed
for 1 min on the Block Test and scanned again to get an I1
image. The Block Test is made of 96 magnets and designed so
that each magnet is centered under the bottom of each well.
After magnet contact, free beads are attracted in the centre of the
bottom of wells, forming a black spot, while the beads blocked
by the biofilm remain in place. Images of each well before (I0)
and after (I1) magnetization were compared with the Biofilm
Control Software. It uses an algorithm combining Fast Fourier
Transform (Press et al., 1992) and precorner detection (Marr and
Hildreth, 1980; Serra, 1988; Jahne, 1997) to estimate the
discrepancy between the two images of a same well, giving a
value named the BioFilm Indice (BFI) ranging from 0 to 30. A
high BFI value corresponds to a high mobility of beads under
magnet action (i.e. control wells) while a low value (b 2)
correspond to a full immobilization of beads (no differences for
a well between its images I0 and I1).
Three experiments with three repeats each (3 wells per slide)
were performed per strain and incubation time. Two strains per
strip were tested, the two resting wells were used as controls
with 200 l of sterile BHI medium containing 6 l ml 1 of
Toner. For each incubation time, the BFI (BFIcontrol
 P. Chavant et al. / Journal of Microbiological Methods 68 (2007) 605612
BFIsample) was determined for the triplicates as well as the mean
and standard deviation. A low BFI value (b 2) means no
biofilm formation and a complete mobility of beads while
higher values correspond to a decreasing mobility of beads due
to the sessile cells. A BFI value higher than 20 corresponds to
the complete immobilization of beads by the sessile cells
forming a biofilm.
To assess the effect of Toner on bacterial growth, 24-h
cultures of each strain with or without Toner were incu-
bated and serially diluted in sterile physiological water (NaCl
90 /00) before plating on BHI agar. Plates were incubated for
24 h at 37 or 30 C according to the strain, and cells were
enumerated.
2.4. Biofilm evaluation with crystal violet staining
This assay, derived from Musk et al. (2005), is based on
the colorimetric measure of crystal violet incorporated by
sessile cells. For each strain, 200 l of IBS were loaded into
the microplate. As a control, 200 l of sterile BHI medium
were used. The plates were prepared in the same way
(strains disposition, temperature and incubation times) than
in the previous BioFilm Ring Test. After each time of
incubation, the medium was removed and wells were
washed three times with 200 l sterile distilled water to
remove non-adherent bacteria. The wells were air dried for
45 min and 200 l per well of a 0.1% (v/v) crystal violet
solution in water (Merck, Fontenay-sous-bois, France) were
added for 45 min. After the staining step of adhered cells,
the wells were washed five times with 300 l of sterile
distilled water to remove excess stain. The dye incorporated
by the adherent cells was solubilised with 200 l of 33% (v/v)
glacial acetic acid (SigmaAldricht, St Quentin Fallavier,
France). The OD of each well was measured at 540 nm
(OD540) by using a microtiter plate reader (iEMS, Thermo
Electron Corporation, Courtaboeuf, France). The absorbance
was measured from the assay OD540 minus control OD540.
The mean values and standard deviations were determined
Fig. 1. Kinetic of biofilm formation by E. coli DH5 ( ); L. monocytogenes EGDe ( ); S. carnosus TM300 () and S. xylosus DSM 20267 ( ) on microplates,
measured with the crystal violet method. Bars represent the standard deviations from three independent experiments with triplicate for each one.
607
for each strain and incubation time. The assay was
performed at least in triplicate with three repeats for each
experiment (3 wells).
2.5. Scanning Electron Microscopy (SEM)
The biofilm in the presence of beads was visualized by
using SEM. To get a greater surface for SEM observations,
biofilms were formed in 6-wells polystyrene plates (Greiner,
Frickenhausen, Germany). The IBS of each strain was mixed
with the Toner at a final concentration of 6 l ml 1 and 6 ml
of this suspension was poured into a well. Six ml of sterile
BHI medium with Toner were poured into one well per plate
as a control. Plates were incubated at 37 C or 30 C
according to the strain. L. monocytogenes EGDe and E.
coli DH5 cultures were observed after 4 and 24-h incuba-
tion while Staphylococci cultures were observed after 2 and
6-h incubation. These times were defined according to
the Biofilm Ring Test and correspond to times before and
after bead immobilization, respectively. After each time of
incubation, the medium was removed and wells were washed
three times on a rotating table (Belly Dancer, Bio-Rad, Ivry-
sur-Seine, France) with 5 ml of sterile physiological water
(90 /00 v/v NaCl) to remove non-adherent bacteria. Sessile
cells were fixed on the support with a solution of 3% (v/v)
glutaraldehyde (Electron Microscopy Science, Washington,
USA) in 0.2 M cacodylate buffered at pH 7.4 (Electron
Microscopy Science) and rinsed in the same buffer. After
postfixation for 1 h with osmic acid vapors (Electron
Microscopy Science), cells were dehydrated using a graded
ethanol series (Prolabo, Fontenay-sous-Bois, France) as
previously described (Chavant et al., 2002). The bottom of
each well were cut, coated with gold in an Emscope SC500
(Elexience, Verrires le Buisson, France) and observed with
a JSM 6060LV SEM (JEOL, Croissy sur seine, France).
Plates were made in duplicate, the first set was used to
observe biofilm formation before the action of magnets, and
the second set was used after magnet application using the
608 P. Chavant et al. / Journal of Microbiological Methods 68 (2007) 605612

Block Test. On the last set, the magnetic field was applied on
the bottom of the wells during all the washing and fixation
steps.
3. Results
3.1. Effects of the BioFilm Toner on bacterial growth
For S. xylosus, the biofilm was visible as a purple ring only
detectable after 24-h culture (OD = 0.65) and reached a
OD = 1.4 after 48-h culture. With the two other strains,
L. monocytogenes and E. coli, a biofilm became detectable only
after 48-h culture with an OD of 0.16 and 0.48, respectively.
With such method important standard deviation were obtained
especially when the biofilm is formed.

Plate enumeration of a 24-h culture of each strain with or
without Toner (magnetic beads solution) revealed no differ-
ences (data not shown) showing that no inhibitory effects on
the bacterial growth could be attributed to the presence of the
Toner.
3.2. Biofilm evaluation with crystal violet staining
During all the experiment, S. carnosus TM300 used as
negative control never formed a biofilm (OD b 0.04) (Fig. 1).
3.3. BioFilm Ring Test
After 10-min incubation, a brown layer was observed to
settle on the bottom of all the wells corresponding to the first
beads sedimented. As evidence that beads were not blocked by
other factors than sessile cells, controls consisting in sterile BHI
medium with Toner were performed. Such control wells always
showed a centred black spot surrounded by a clear zone after
magnetization due to the beads attracted and concentrated by
the magnet (Fig. 2 wells 1, 5, 9 and 13). This highlights the fact

Fig. 2. Kinetic of biofilm formation on polystyrene microplates by L. monocytogenes EGDe (wells 2 to 4), E. coli DH5 (wells 6 to 8), S. xylosus DSM 20267 (wells
10 to 12) and S. carnosus TM300 (wells 14 to 16) with the BioFilm Ring Test after different incubation times. Images were scanned with the Plate Reader before and
after application on the Block Test. Controls with only BHI medium and Toner are represented by wells 1, 5, 9 and 13.
 P. Chavant et al. / Journal of Microbiological Methods 68 (2007) 605612
Fig. 3. Biofilm formation by L. monocytogenes EGDe ( ), E. coli DH5 ( ), S. xylosus DSM 20267 ( ) and S. carnosus TM300 () obtained with the BioFilm
Control image analysis software. Results are expressed as BFI (BioFilm Indice) according to the incubation time. Bars represent the standard deviation obtained
from the three independent experiments with triplicate for each one.
that beads settled on the surface and kept their potential of
magnetization during all the experiment. Thus, shape or
intensity variations of the black spot in wells containing
bacteria may be attributed only to interactions with these
microorganisms. The biofilm-negative strain S. carnosus also
displayed black spots during all the experiment, showing that
beads were always attracted by magnets and not retained by
cells (Fig. 2, wells 14, 15, 16). For the three others strains, black
spots were only visualized at the beginning of the experiment.
Further, due to the adhesion of cells, their growth and the
formation of bacterial mats or aggregates, the magnet attractive
forces became not stronger enough to overcome forces retaining
beads in the biofilm and to attract them in the centre of the wells,
resulting in the fading of the spot.
Biofilm assays of L. monocytogenes (Fig. 2, wells 2, 3, 4)
showed a black spot within the first 6-h culture. After 8-
h incubation, a slight spot appeared, surrounded by a narrow
clear zone, meaning that only the nearest beads were attracted.
In 24-h cultures, no black spot appeared anymore. Biofilm
assays with E. coli (Fig. 2, wells 6, 7, 8) revealed the black
spot surrounded by a large clear zone within the first 4-
h incubation. Afterwards, the black spots became diffuse and
completely disappeared as soon as 8-h incubation. Among the
four strains, S. xylosus was the earliest and formed a biofilm
after 4-h.
The kinetic of biofilm formation with the Biofilm Ring
Test is showed in Fig. 3. The image analysis applied on the
BioFilm Ring Test gives an indice named the BFI (BioFilm
Indice) which reflects the potential mobility of beads under the
magnetic field applied. Displayed in function of the time, the
BFI (BFIcontrols BFIsamples) gives a profile in concordance
with the increasing bead immobilization and thus with the
increasing resistance forces generated by the biofilm. The
earliest times (BFI b 2) corresponds to the complete bead
mobility due to the absence of a biofilm. Further, the
increasing BFI reflects the progressive immobilization of
the beads during biofilm formation until reaching a plateau
(N 20) corresponding to a complete immobilization of beads.
609
This plateau means that the surface of the well is strongly or
completely covered by cellular aggregates, mat or three-
dimensional biofilm structures which prevent any bead
mobility as shown by SEM. Here, the BFI of S. carnosus
never increased while the BFI values of the three other
strains revealed very rapid immobilization of beads after
various incubation times. The plateau (value 20) was
reached after 4-h for S. xylosus, 8-h for E. coli and between
8 and 24-h cultures for L. monocytogenes. Moreover, low
standard deviation were obtained with such method during all
the experiment.
3.4. SEM observations
Biofilm formation on 6-wells polystyrene plates for
L. monocytogenes, E. coli, S. xylosus and S. carnosus, ac-
cording to the BioFilm Ring Test, were observed by SEM
(Fig. 4). For each strains, two incubation times before and after
reaching the BFI plateau and before and after application on
the Block Test were selected from the previous BioFilm Ring
Test experiments.
Beads appeared spherical, dispersed and uniform in their
size (1 m). For all the strains the observation of the black
spot, as long as it was visible after application on the Block
Test, revealed a mass of beads and no bacteria (see data from
S. carnosus Fig. 4E). After 2-h incubation with S. xylosus,
only small aggregates containing few cells were observed on
the surface of wells (Fig. 4A, B). At this time, before mag-
netization, some beads not removed by the washing steps
remained on the surface and were generally in contact with
bacteria (Fig. 4A). After magnetization, all the free beads were
attracted by magnet and/or removed by SEM washing steps
and only some small bacterial aggregates were always ob-
served on the surface, in contact with few beads (Fig. 4B). In
6-h cultures, the BioFilm Ring Test did not show a black spot
anymore and a layer of S. xylosus cells covering the surface
was observed before or after application on the Block Test
(Fig. 4C, D). Many beads remained embedded into the biofilm,
610 P. Chavant et al. / Journal of Microbiological Methods 68 (2007) 605612

Fig. 4. SEM observations of polystyrene 6-wells microtiter plate surface inoculated according to the BioFilm Ring Test. 2-h (A, B) and 6-h (C, D) cultures of
S. xylosus before (A, C) and after (B, D) application on the Block Test. Beads concentrated in the centre of the well (black spot) of a S. carnosus 6-h culture after
application on the Block Test (E) and detail of a very small and scattered cell aggregate in the clear area surrounding the black spot (F). 4-h (G) and 24-h (H) cultures of
L. monocytogenes after application on the Block Test, showing aggregates and three-dimensional shapes with embedded beads, respectively. Bacterial cells (c);
magnetic beads (mb).

entrapped into a 3-D structure strong enough to overcome the
magnetic forces applied on them, resulting in the fading of the
black spot.
SEM observations of the biofilm-negative strain S. carnosus
revealed that this microorganism never formed a biofilm
and only scattered groups of three or four bacteria could be
observed (Fig. 4F). Then after magnetization, the beads
were all grouped in the centre of the well (Fig. 4E). For
L. monocytogenes, the 4-h cultures submitted to magnet action
showed some cell aggregates but no continuous mono-layer
(Fig. 4G). After 24-h incubation, a thick biofilm with a three-
dimensional architecture containing embedded beads was
visible (Fig. 4H). Same observations were done with E. coli
(data not shown).
 P. Chavant et al. / Journal of Microbiological Methods 68 (2007) 605612
4. Discussion
Bacterial adhesion and biofilm formation are very important
concepts in the area of medicine, industry and environment, that is
why the development of methods to measure the capacity of
various bacterial strains to adhere and to form such structures
appears of great interest. To date, a limited number of methods are
used to detect the biofilm formation from one microorganism. The
conventional methods are enumeration by plating or microscopy.
However, plating is a laborious technique requiring that bacteria
are released first and do not detect cells that are viable but not
culturable or which died during the process. Similarly, use of
microscopy techniques (epifluorescence, laser-scanning confocal,
transmission electron and scanning electron microscopy) are also
laborious methods, often requiring numerous steps for sample
preparation and the observation of many fields to avoid observer
error. Thus, these conventional methods are not adapted to high
throughput screening of a great number of bacterial strains or to
test one microorganism under different environmental factors.
Today, the mainly recognised and developed technique allowing
such screening consists in a colorimetric method based on the
staining of sessile cells by crystal violet for example. But this
staining technique is rather long and time consuming and often
gives high variations for a same result (Vesterlund et al., 2005).
From these observations, the development of a new method,
faster, reliable and reproducible, to evaluate the ability of
microorganisms to adhere and to form biofilms on a surface
appeared of great interest.
In this study, we present a new device named the BioFilm
Ring Test to evaluate the capacity of different strains to form
biofilms in BHI medium on polystyrene surface. This test is
based on the concept of immobilization of beads by sessile
bacteria forming aggregates and/or a mat with strength enough
to overcome the magnetic attraction forces applied on them.
Thus, in the absence of sessile cells, all the beads are gathered
by a magnet centered under the well and designed a black spot
easily detectable. In order to assess the performance and the
reliability of the BioFilm Ring Test, this new device was
compared to the crystal violet assay with a minimum panel of
four strains combining different bacterial phenotypes (bacillus
and cocci, Gram-positive or negative, high polymer producers
or not,). SEM was also performed to visualize biofilm and
bead interactions after different times of incubation.
Results obtained with both methods led to similar trends and
revealed that S. carnosus was biofilm-negative while the three
other strains were positive and detected in the same chronology
(S. xylosus first, then E.coli and lastly L. monocytogenes). Such
results are in agreement with the literature describing S.
carnosus TM300 as a strain devoided of the icaA gene encoding
the Polysaccharide Intercellular Adhesin involved in intercel-
lular adhesion and associated with strong biofilm formers
(Cramton et al., 1999). Among the three biofilm-positive strains
detected, S. xylosus is well known to produce a lot of
exopolymers (Planchon et al., 2006). So, it is not surprising to
detect rapidly its thick and mucous biofilm containing a great
number of embedded cells in comparison to E. coli DH5 or
L. monocytogenes EGDe a weakest polymer producer (Wirta-
611
nen and Mattila-Sandholm, 1993; Midelet and Carpentier,
2002). Therefore, we note that a time scale ranging from 24 h to
48 h is required with the staining method to detect a biofilm, as
usually related in the literature (Stepanovic et al., 2000;
Djordjevic et al., 2002) while these data are collected in less
than 24-h with the BioFilm Ring Test.
When comparing the BioFilm Ring Test with the crystal
violet assay, it is important to consider the inherent advantages
and disadvantages of each techniques. The crystal violet is a
well established method allowing an indirect quantification of
the sessile cell number. Its major disadvantage is the need of
many non standardized manipulations (washing steps, staining,
destaining, drying) which may lead to some important standard
deviations as observed in our study. On the other hand, the
BioFilm Ring Test requires very few manipulations after the
initial bacterial inoculation (no washing and staining steps).
Results are directly read by transferring the strips on the Plate
Reader after one minute exposure on the Block Test. So, this test
is less time consuming and need less handling in comparison
with the crystal violet method ensuring a better reproducibility
and lowest standard deviation. Moreover, this method allows an
early detection of the biofilm-positive strains, at least twice
faster than with the crystal violet assay. It is clear that the
addition of Toner increases the surface area into the microtiter
well and might promote biofilm formation depending on the
bacteria affinity. However, SEM studies shown (i) no bacterial
aggregates on beads during the early stages but rather bacterial
cells first adhered on the well surface with beads on, and (ii) no
adherent bacteria on the beads attracted by the magnet, forming
the black spot area. This strongly suggests that bacteria
preferentially adhered to the polystyrene surface and not first
on the beads. It is also well known that the first steps of
adhesion are support-dependent (Toutain et al., 2002) so the
technique we described here, validated on polystyrene (i.e.
hydrophobic surface) will be further extended by using others
supports, either hydrophobic (polycarbonates) or hydrophilic
(glass,). The culture medium is also a critical parameter for
adhesion efficacy according to the bacterial species (Moltz and
Martin, 2005; Planchon et al., 2006), so new tests are planned
with different culture media and microorganisms.
In conclusion, the BioFilm Ring Test may be used as a
rapid, reproducible and easy-handling method to study the
kinetic of bacterial biofilm formation in BHI medium. This
assay can be implemented for high throughput screening of a
great number of bacterial strains or mutants by using different
growth parameters (pH, temperature, culture medium) or for the
rapid screening of anti-biofilm molecules (soluble or grafted on
surface). In this way, the throughput of this new device will be
dramatically increased by its future automatisation.
Acknowledgement
We thank I. Lebert, N. Garrel, B. Duclos (Institut National de
la Recherche Agronomique, INRA site de Theix, France) and J.
Groelly (BioFilm Control SAS) for their technical assistance.
We thank Mr. N. Bara and the C.L.E. team (plastic parts), and
Mr Barthelemy (IMSA) for their tireless support.
612 P. Chavant et al. / Journal of Microbiological Methods 68 (2007) 605612
This work was supported in part by the ANVAR (Agence
Nationale pour la Valorisation de la Recherche) and by the
BioFilm Control SAS.
The Biofilm Ring Test concept is under patent WO 2005/
090944 A1 and PCT/FR05/00427 (T. Bernardi, 29 September
2005, French patent office).
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