Peptone
yeast
extract
with
hemin
and
Vitamin
K
(Wilkins
Chalgren
broth,
Oxoid
Ltd,
UK)
Phosphate
buffered
saline
(PBS)
Sulfamethoxazole
Trimethoprim
Carboxymethylose
Procedure:
Bacterial
preparation
P.
gingivalis
and
F.
nucleatum
are
grown
in
peptone
yeast
extract
containing
hemin
and
vitamin
K
in
an
anaerobic
chamber
followed
by
three
washes
in
PBS.
Bacterial
concentration
is
spectrophotometrically
standardized
to
OD650nm
=
0.1
for
P.
gingivalis
(1010
bacteria/mL),
and
OD660nm
=
0.26
for
F.
nucleatum
(109
bacteria/mL).
Procedure:
Bacterial
infection
and
harvesting
maxillae
Mice
are
pre-treated
with
sulfamethoxazole/trimethoprim
(0.08%
and
0.016%,
respectively)
in
drinking
water
for
10
days,
followed
by
a
three
day
wash
out
(no
antibiotics).
Treated
mice
are
infected
with
400
µL
containing
109
bacteria/mL
of
each
pathogen
at
days
0,
2,
and
4,
using
2%
carboxymethycellulose
in
PBS.
Control
mice
receive
PBS
only.
Mice
are
euthanized
at
42
days
post-infection
and
maxillae
harvested.
Procedure:
microCT
analysis
Samples
are
placed
in
a
cylindrical
sample
holder
and
approximately
200
microtomographic
slices
are
obtained
(12
µm
increments)
covering
the
entire
bucco-palatal
width
of
each
hemi-jaw.
Maxillary
hemi-jaws
are
analyzed
by
compact
fan-beam-type
computerized
tomography
(microCT).
For
calculation
of
bone
volume
in
control
and
treated
mice,
a
reference
line
is
set
throughout
the
microtomographic
slices
at
a
distance
from
the
cemento-enamel
junction,
chosen
to
be
below
the
horizontal
zone
of
destruction
in
treated
mice.
Bone
volumes
are
recorded
for
control
and
treated
mice.
Data
collected
by
investigator
Maxillary
jaw
alveolar
bone
volume:
uninfected,
control
volume
Maxillary
jaw
alveolar
bone
volume:
infected,
residual
volume