Chapter 48

Biofilm Formation in the 96-Well Microtiter Plate

Barbara M. Coffey and Gregory G. Anderson

Abstract
The microtiter plate (also called 96-well plate) assay for studying biofilm formation is a method which
allows for the observation of bacterial adherence to an abiotic surface. In this assay, bacteria are incubated
in vinyl “U”-bottom or other types of 96-well microtiter plates. Following incubation, planktonic bacteria
are rinsed away, and the remaining adherent bacteria (biofilms) are stained with crystal violet dye, thus
allowing visualization of the biofilm. If quantitation is desired, the stained biofilms are solubilized and
transferred to a 96-well optically clear flat-bottom plate for measurement by spectrophotometry.

Key words Adhesion, Bacteria, Biofilm, Crystal violet, Microtiter, Pseudomonas, Surface-adhered,
96-well

1 Introduction

Biofilm formation occurs when bacteria switch from a planktonic

(free-swimming) state to a surface-attached state. Pseudomonas
aeruginosa biofilms can be found on a wide range of natural and man-
made surfaces, including rocks, oil pipelines, medical catheters [1],
and human tissue. To study biofilms, a number of laboratory-based
biofilm formation models have been developed. The method of
studying biofilm formation on plastic tissue culture plates was first
described in the 1970s, and the use of 96-well tissue culture and
microtiter plates was introduced in the 1980s [2, 3]. This assay
tests the ability of bacteria to adhere to the plastic surface of a
microtiter plate under the temperature and nutritional conditions
determined by the experimenter. The method was first published
in 1996 by Genevaux et al. as a way to perform rapid screens for
Escherichia coli surface attachment [4]. Two years later, the assay
was adapted for Pseudomonas by O’Toole et al. to support genetic
studies of biofilm formation. The protocol continues to be used
widely in studies of microbial biofilms [5, 6].
Today, there are a number of methods for growing and analyzing
biofilms in a laboratory setting, including assays for air–liquid

Alain Filloux and Juan-Luis Ramos (eds.), Pseudomonas Methods and Protocols, Methods in Molecular Biology,
vol. 1149, DOI 10.1007/978-1-4939-0473-0_48, © Springer Science+Business Media New York 2014

631
632 Barbara M. Coffey and Gregory G. Anderson

interface biofilms, colony biofilms, drip-fed biofilms, and flow-cell

biofilms [7–9]. The advantages of the microtiter plate assay
described here are its simplicity, the use of basic lab materials, the
adaptability to small or large numbers of samples, and the variety
of samples that can be tested in a single assay. This protocol has
been used successfully in biology classroom settings at the high
school level [10]. The microtiter plate assay is particularly useful
for studying the early steps in biofilm formation, such as initial
surface attachment. To examine mature biofilms, this method
could be complemented with a flow system.
Various modifications have been implemented for monitoring
specific activities. For instance, in a system known as the Calgary
Biofilm Device, designed especially for antibiotic susceptibility
testing, biofilms are grown on the surface of 96 pegs submerged in
liquid medium [11]. Additionally, there are several effective stains
used for biofilm evaluation, including crystal violet (described
below), safranin, and Alamar blue [12]. In the last few years,
safranin staining has been described in several biofilm studies of
Gram-positive bacteria [13–15].
In the microtiter plate biofilm protocol, bacteria are first
grown planktonically to stationary phase and then subcultured
into fresh medium for the assay. 100 μL aliquots of the freshly
inoculated samples are transferred to a microtiter plate and incu-
bated. Incubation temperature is commonly 37 °C, for a length of
time ranging from as little as 30 min to as much as 72 h. Assay
medium, incubation temperature, and length of incubation may be
varied according to the desired experimental parameters. Following
incubation, the microtiter plate is rinsed in water. The water
washes away non-adherent bacteria, but any biofilm that has
formed will remain attached to the microtiter plate. The wells are
then filled with 125 μL of 0.1 % crystal violet and allowed to sit for
10 min. The crystal violet will stain the biofilm but not the vinyl
microtiter plate.
Crystal violet belongs to the family of triphenylmethane dyes,
which bind to the bacterial cellular components by ionic interac-
tions. It has been shown to bind to DNA [16], proteins [17], and
apparently polysaccharides, including biofilm polysaccharides
[18]. When the crystal violet is rinsed away, biofilm formation
will be visible as a ring around the inside of the well (Fig. 1). If
additional quantitation of the biofilm is desired, the stained bio-
film should be allowed to dry for several hours. The biofilm can
then be solubilized, transferred to an optically clear flat-bottom
96-well plate, and measured by spectrophotometry. This quanti-
tation method does not necessarily correlate directly with the
number of viable bacterial cells in the biofilm, since the matrix of
extracellular DNA, polysaccharides, and proteins is also stained by
crystal violet. Additionally, crystal violet kills bacteria, making this
staining method incompatible with further analysis of viable cells.
 Microtiter Plate Assay 633

Fig. 1 Rings of crystal violet-stained biofilm are visible in the wells of a vinyl
microtiter plate (indicated by arrow). (a) Looking into the wells from the top. (b)
Plate is inverted. Rings of biofilm are visible in bottom wells, with no biofilm
formation visible in top wells

If viable cell counts are required, a different method must be

used to remove the biofilm from the surface of the microtiter
plate and transfer it to another growth medium. Finally, findings
discovered using microtiter plate assays should be confirmed by
microscopy or another method.

2 Materials

Store all materials and solutions at room temperature.

2.1 Biofilm Assay 1. 96-well microtiter plate, not tissue culture treated (see Note 1).
2. Four flat containers of sufficient size to hold 96-well plate sub-
merged in liquid (see Note 2).
3. 0.1 % crystal violet solution in water (see Notes 3–5).
4. Pipetter and 200 μL tips (see Note 6).
5. Bacteria of interest.
6. Media appropriate for bacterial growth and/or variations of
media as desired for experimental purposes (see Note 7).
7. Lab mat or paper towels (see Note 8).
8. Lab coat and gloves.
9. Container for liquid waste (see Note 9).
634 Barbara M. Coffey and Gregory G. Anderson

10. Container for dry waste (e.g., pipette tips).

11. Funnel (see Note 10).
12. Trough or empty tip box for pipetting with multichannel
pipetter.
13. 30 % glacial acetic acid solution for solubilizing biofilm
(see Note 11).
14. 1.5 mL microcentrifuge tubes.
15. Water: Non-sterile tap water is fine.

2.2 Biofilm Some of the most commonly used media for biofilm assays are LB,
Assay Media Mueller–Hinton broth (MHB), M9 minimal medium, and M63
minimal medium supplemented with casamino acids and glucose
[19]. LB and MHB are nutrient-rich general-purpose media for
growing bacteria; they provide a nutritional environment that
allows for P. aeruginosa biofilm growth. MHB is often used in
experiments on antibiotic susceptibility testing. However, when
the focus of an assay is to determine the effect of a particular nutri-
ent on biofilm formation, it is necessary to use a chemically defined
medium in which the molar concentrations of the nutrient(s) in
question can be controlled. Media which can be manipulated in
this manner include M9, M63 with 0.4 % arginine [20], N-minimal
medium [21], and B2 [22]. A number of other media can be used,
as desired.
1. M9: Per liter of water, 64 g Na2HPO4, 15 g KH2PO4, 2.5 g
NaCl, 5.0 g NH4Cl, 2 mL of 1 M MgSO4, 0.1 mL of 1 M
CaCl2, and 20 mL of 20 % glucose solution.
2. M63: Per liter of water, 13.6 g KH2PO4, 2.0 g (NH4)2SO4,
0.2 g MgSO4, 0.5 mg FeSO4, glucose (0.2 %), and casamino
acids (0.5 %) [5]. The addition of arginine to a final concen-
tration of 0.4 % enhances P. aeruginosa biofilm formation
[23].
3. N-minimal: 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K 2SO4,
1 mM KH2PO4, 0.1 M Tris–HCl, 0.1 % casamino acids,
38 mM glycerol, 200 μM bis-Tris [21].
4. B2: 0.1 M HEPES pH 7, 7 mM ammonium chloride, 20 mM
sodium succinate pH 6.7, 2 mM magnesium sulfate, 10 μM
iron sulfate, 1,600 μM phosphate buffer pH 7.2, 1.62 μM
manganese sulfate, 2.45 μM calcium chloride, 13.91 μM zinc
chloride, 4.69 μM boric acid, 0.67 μM cobalt chloride.

2.3 Quantitation 1. 30 % glacial acetic acid in water (see Note 11).

2. 96-well optically clear, flat-bottom plate (can be non-sterile).
3. Spectrophotometer equipped with plate reading capability.
 Microtiter Plate Assay 635

3 Methods

3.1 Preparation 1. See Figs. 2 and 3 for suggested layout of materials.

of Biofilm Assay Work 2. Bench area to be used for biofilm staining should be lined with
Area lab mat or paper towels. A space approximately 2 ft (60 cm) wide
is sufficient. It is a good idea to set up a piece of cardboard or

Fig. 2 Suggested benchtop layout for microtiter plate biofilm assay. Waste liquid
waste container, CV bottle containing 0.1 % crystal violet, D disposal container
for used pipette tips, P pipetter (single or multichannel), 1 tray 1 (waste), 2 tray 2
(crystal violet rinse 1), 3 tray 3 (crystal violet rinse 2), 4 tray 4 (rinse), E empty tip
box or trough, T pipette tips, Blot extra layer of lab mat or paper towel

Fig. 3 Example of benchtop set up for microtiter plate biofilm assay. Note the
location of materials portrayed in Fig. 2. Arrangement of materials is adaptable to
fit a variety of different laboratory configurations
636 Barbara M. Coffey and Gregory G. Anderson

another material as a shield to prevent neighboring bench

space from being splashed with crystal violet stain.
3. Arrange the following materials on lined bench top: Four trays,
bottle of 0.1 % crystal violet solution, pipetter, pipette tips,
container for liquid waste, funnel, container for used pipette
tips, trough, or empty tip box (see Fig. 2, Note 8).
4. Fill trays 2, 3, and 4 with enough water to submerge microtiter
plate. This water can be from the tap and need not be sterile.
Water can be left in trays and reused for several assays. When
the water in trays 2 and 3 becomes heavily stained with crystal
violet, it should be dumped into the liquid waste containers
and replaced with fresh water. The water in tray 4 should
remain free of crystal violet but will become cloudy with bacteria,
at which time it should be dumped into the waste container
(see Note 9) and replaced with fresh water.

3.2 Preparing 1. Inoculate bacteria into 3–5 mL appropriate growth medium in

Bacterial Cultures sterile culture tubes and grow to stationary phase. Typically,
bacteria are grown overnight at 37 °C with shaking. If a large
number of strains are to be assayed at once, such as when
screening a mutant library, it may be desirable to grow cultures
in 96-well plates instead of culture tubes.
2. Dilute the stationary-phase cultures 1:100 into fresh assay
medium, which may vary according to the desired experimen-
tal parameters (see Note 12). It is recommended to assay all
samples in triplicate (or more) so that mean values can be
obtained and statistical analysis performed (see Note 13).
3. Aliquot 100 μL of diluted cultures into 96-well microtiter
plate. Be sure to include a blank well containing uninoculated
medium as a control.
4. Cover plate (see Note 14) and incubate at appropriate
temperature for desired length of time (see Notes 15 and 16).
An incubation of 8–10 h is suggested for most purposes [5].

3.3 Staining 1. All the remaining steps are completed at room temperature.
of Biofilms 2. Following desired incubation time, remove planktonic cells by
inverting 96-well plate over waste tray (tray 1, see Fig. 2) and
shaking it gently.
3. Rinse the microtiter plate by submerging it in clear water in the
rinsing tray (tray 4). While the plate is submerged, gently rub
the surface of the plate with your gloved fingers to release bub-
bles and ensure that water enters all wells. After rubbing the
entire surface of the plate, lift it out of the water, invert it, and
shake out excess water back into the tray. Repeat this step in
the same tray so that plate is rinsed two times.
4. Turn the plate face down, and pat it firmly on lab mat or paper
towel to remove as much water as possible.
 Microtiter Plate Assay 637

5. Pipette 125 μL 0.1 % crystal violet solution into wells. This

volume ensures that the stain will cover the biofilm.
6. Let sit for 10 min.
7. Invert plate over waste tray (tray 1) and shake gently to remove
liquid.
8. Submerge plate into tray 2, and rub the entire surface of the
plate to ensure that water enters all wells. Remove plate from
water, invert, and shake to remove liquid. Repeat so that plate
is rinsed twice in tray 2.
9. Submerge plate into tray 3 and rinse twice as described in the
previous step.
10. Invert plate over tray 3 to remove excess water.
11. With plate face down, pat firmly on lab mat or paper towel to
remove as much water as possible.
12. At this point, you may be able to see purple rings where bio-
films have formed at the air–liquid interface on the inner sur-
face of the plastic wells (Fig. 1). Also, there may be biofilm on
the bottom of the wells. It is important to note that crystal
violet stains any substance that may be adhered to the plastic
surface, including extracellular polymers or crystallized
substances.
13. Allow the plate to dry for several hours, until all excess water is
evaporated, prior to proceeding with quantitation.
14. If there is no visible biofilm formation in any of your samples,
there are several variables you may need to adjust. Media compo-
sition, length of incubation, and incubation temperature are of
primary importance. We have observed consistent P. aeruginosa
biofilm formation (as shown in Fig. 1) in M63 medium with
0.4 % arginine, given an incubation of 24 h at 37 °C. If your assay
does not produce biofilm formation under these conditions,
check to make sure that the assay samples were indeed inocu-
lated and that the assay medium did not evaporate during the
incubation time.

3.4 Quantitation 1. Allow stained biofilm plate to air-dry for several hours or
of Biofilms overnight.
2. Pipette 150 μL 30 % acetic acid into each well. This will solubilize
the biofilm (see Note 11).
3. Allow to sit for 10 min.
4. Pipette up and down to assure that the biofilm is well solubi-
lized, and then transfer 125 μL of each sample to a 96-well
optically clear, flat-bottom plate.
5. Measure optical density of all samples in plate at 550 nm
(see Note 17). Data may be graphed as shown in Fig. 4.
638 Barbara M. Coffey and Gregory G. Anderson

Fig. 4 Example of data that may be obtained by using spectrophotometry to quantitate biofilm formation.
The bar graph at left is a graphical representation of the data shown in the table on the right. Numbers
represent absorbance value

4 Notes

1. Several types of microtiter plates are available. We most often

use vinyl U-bottom plates; however, flat- or V-bottom plates
made of other materials (e.g., polystyrene or polypropylene)
can also be used. Adhesion will vary depending on the proper-
ties of the surface and the P. aeruginosa strain (e.g., mucoid
versus non-mucoid) [24].
2. Plastic food storage containers or large pipette tip boxes work
well for this purpose. The container need to be just deep
enough to submerge a 96-well plate. These containers will
become heavily stained with crystal violet and should be used
only for biofilm staining.
3. It is important to be aware of the staining properties of the
crystal violet dye used in this assay. It is recommended that
materials and a small portion of lab bench space be dedicated
exclusively to this process.
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.
5. Crystal violet is classified in some countries as a hazardous sub-
stance. The use of a chemical hood while working with crystal
violet is advised. Refer to your institution’s chemical safety
protocols for more information.
6. Multichannel pipetters greatly increase the time efficiency of
these assays.
 Microtiter Plate Assay 639

7. If the purpose of the experiment is to observe P. aeruginosa

biofilm formation in a particular liquid substance (e.g., mouth-
wash, tap water, soft drinks, liquid detergent), these liquids
should be used in place of the assay media described here.
Thus, the stationary-phase bacteria culture would be diluted
1:100 into the substance of interest. For the first attempt at
growing biofilms in liquids other than bacterial growth
medium, it may be useful to include a range of dilutions, such
as 1:10, 1:50, 1:100, and 1:500. This will allow a determina-
tion of the optimal dilution to use in future experiments so as
to yield observable results.
8. To help prevent unwanted stain splatters in the lab, it is best to
have all materials set up and ready to use before you begin
biofilm staining so that you do not have to walk around the lab
gathering supplies (e.g., timer, extra pipette tips, paper towels,
gloves) while you are in the middle of the staining procedure.
9. Liquid waste should be placed in an autoclave-safe (e.g., glass)
container. It is recommended that the liquid waste be autoclaved
before disposal. It is best to autoclave when the container is no
more than 2/3 full so that crystal violet waste does not spill
over the top of the container due to pressure changes in the
autoclave. Check with your institution’s chemical safety protocol
for proper disposal procedure.
10. A small funnel may be needed to transfer liquid waste from
trays into waste container if the waste container is a narrow-
neck bottle. This funnel will become stained with crystal violet,
and so it may be desirable to dedicate a funnel specifically to
biofilm assays.
11. A solution of ethanol–acetone (70:30) or 95 % ethanol may be
used to solubilize stained biofilm. These solutions are particularly
effective on biofilms formed by Gram-positive species [25].
12. Prior to diluting cultures, it is suggested that the density of the
original cultures be adjusted to OD600 = 0.05. This will com-
pensate for differences in cell densities of the original cultures
that could potentially affect the amount of biofilm formed
during the assay.
13. Before beginning to aliquot samples, particularly if working
with a large number, it is a good idea to plan how the samples
will be distributed in the 96-well plate and to make a written
note of the layout. Permanent marker can be used to label rows
and/or columns on the biofilm assay plate prior to adding
samples. Labeling should not be done, however, on samples in
an optically clear plate being used for spectrophotometry, as ink
may interfere with readings.
14. The plate may be covered with a lid designed for 96-well plates.
Placing the covered plate in a disposable plastic storage
640 Barbara M. Coffey and Gregory G. Anderson

container (such as those used for food) can reduce evaporation

of media in the incubation chamber. Plate lids and storage
containers may be reused by rinsing between uses with 70 %
ethanol and allowing to air-dry.
15. How is incubation time determined? If the desired results of the
assay are simply to determine whether or not a certain sample
forms biofilm, without the need to define how long it takes or
to quantitate results, then an overnight incubation is typically
sufficient for P. aeruginosa to form biofilms. However, if the
incubation is too long, the small volume of medium in the
wells will become depleted of nutrients. When nutrients are
unavailable, bacteria will begin to die, after which point no
additional biofilm will form, and cellular debris may aggregate
in the microtiter plate and produce confusing results. Studies
have shown maximum biofilm formation in as little as 8–10 h [5]
or as much as 72 h [23], depending on the Pseudomonas strain
being tested, the nutrient content of the assay medium, and
the type of microtiter plate. In some cases, the experimenter
may wish to shorten the incubation time if, for example, the
question is to see which strains or conditions produce biofilm
most rapidly. In such experiments, the incubation time may be
as little as 30 min, although it may take several assays and
adjustments to the incubation time to optimize the results.
16. Depending on desired experimental parameters, media may or
may not be changed at intervals throughout the incubation
period [26]. For instance, many studies have used the microtiter
plate assay to investigate the effect of antibiotics or other anti-
microbial agents on P. aeruginosa biofilms.
17. Optical density of crystal violet-stained samples may be measured
in a range of 500–600 nm [7].

Acknowledgements

This work is supported by RSFG (Indiana University Purdue

University Indianapolis) and PRF (Purdue University) to Gregory
G. Anderson.

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