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2% sucrose in MatTek culture plates b at 37°C and 5%
CO 2
. The culture medium was replaced daily with
fresh 37°C medium over a 3-day culture period. The
biofilms were stained to show the bacterial cells in
green and the slime matrix in blue. The images were
constructed using Imaris software. c
b
MatTek Corp; Ashland, MA.
c
Bitplane, AG; Zurich, Switzerland.
Diffusion Chamber
A previously developed method 22-24 was used to
assess the effect of power brushing on the diffusion
of fluoride through biofilms. A specially designed 2-
chamber diffusion chamber was fabricated from
polycarbonate (Figure 2).
The 2 chambers were separated by a cellulose
ester membrane. By growing biofilms on the membrane
and measuring how quickly fluoride passed
from one chamber to the other, we could measure
the effect of power brushing on delivering fluoride
through the biofilm-colonized membrane. The membrane
filter holder was easily inserted into the diffusion
chamber allowing rapid replacement with new
biofilm-colonized filters for repeated measurements.
First, the biofilm-side chamber (right-hand chamber)
was gently filled with 1100 ppm sodium fluoride
solution in phosphate buffer salts (PBS). The
measurement-side chamber contained PBS with
approximately 0.5 ppm fluoride. By starting with a
small concentration of fluoride in the measurement
chamber, the stabilization time of the electrode was
decreased. Thus, at time zero, the measurement-side
chamber had 0.5 ppm fluoride, whereas the biofilmside
chamber had 1100 ppm. The filling process took
approximately 5 seconds. The appearance of fluoride
in the measurement chamber, after traveling through
the biofilm and the membrane, was measured by an
ion-selective fluoride electrode. The measurementside
chamber was mixed vigorously with a magnetic
stir bar. Over the 4-minute monitoring period, the
concentration in the biofilm-side chamber never fell
to less than 1050 ppm, so it was assumed that the
concentration gradient driving the fluoride flux was
constant.
Biofilm Growth for Diffusion Measurements
Biofilms were grown on the cellulose ester
membranes from S mutans UA159 by immersing a
filter holder, with an attached filter, in each well of a
6-well plate with 5 mL of BHI and 2% sucrose.
Because the filter assemblies were slightly buoyant,
they floated, so that the filter surface was level with
Figure 3—The dental plaque bacterial biofilms were composed of dense clusters of bacteria attached to the surface, enveloped in a protective
extracellular polymeric slime (EPS) matrix
A. Three-dimensional (3-D) confocal image of a natural dental
biofilm grown in vivo on an enamel surface positioned in the region
of the premolars and molars for 3 days. Fluorescent in situ hybridization
was used to identify specific bacteria. All bacteria were stained
red with a universal probe. Streptococci were stained yellow. The
enveloping slime matrix is shown as blue. The EPS in the foreground
was made transparent using the Imaris 3-D rendering software
(Bitplane) allowing the bacteria inside to be visible. In some places,
channels (white arrow) protruded all the way through the biofilm to
the enamel underneath. Scale: major grid divisions are 20 µm.
B. 3-D confocal image of a laboratory-grown Streptococcus
mutans biofilm showing individual cocci (green) enveloped in a
slime matrix (blue). Scale: major grid divisions are 10 µm.
18 Compendium / September 2007 Vol. 28, No. 9 (Suppl 1)