APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
Oct.
1986,
p.
971-973
0099-2240/86/100971-03$02.00/0
Copyright
C
1986,
American
Society
for
Microbiology
Vol.
52,
No.
4
Quantitative
Method
for
Measurement
of
Aerotolerance
of
Bacteria
and
Its
Application
to
Oral
Indigenous
Anaerobes
HIROKO
E.
KIKUCHI*
AND
TAKESHI
SUZUKI
Department
of
Oral
Microbiology,
School
of
Dentistry,
Hokkaido
University,
Sapporo,
Japan
Received
31
March
1986/Accepted
11
July
1986
An
index
which
expressed
the
aerobic
to
anaerobic
potential
was
made
for
bacteria
with
intermediate
tolerance
for
oxygen.
One
method
used
for
this
analysis
was
measurement
of
the
relative
bacterial
growth
ratio.
The
other
method
was
based
on
the
pattern
of
the
absorbancy
versus
depth
plot.
The
index
was
applied
to
oral
indigenous
anaerobes.
Brock
(1)
classified
bacteria
into
five
groups
as
follows:
(i)
obligate
aerobes,
(ii)
facultative
anaerobes,
(iii)
aerotolerant
anaerobes,
(iv)
obligate
anaerobes,
and
(v)
microaerophilic
organisms.
He
described
facultative
anaerobes
as
bacteria
that
can
grow
with
and
without
oxygen
and
aerotolerant
anaerobes
as
bacteria
that
can
grow
in
the
absence
of
oxygen.
However,
the
group
definitions
of
Brock
are
de-
scriptive,
and
classifying
every
bacterium
is
practically
difficult.
It
is
also
difficult
to
compare
the
resistance
for
the
oxygen
effect
from
one
bacterium
to
others.
The
purpose
of
the
present
study
was
to
provide
quanti-
tative
measurements
of
the
aerotolerance
of
microorga-
nisms.
Two
methods
were
used;
one
involved
the
shaking
of
a
liquid
culture
and
the
other
involved
an
agar-mixed
culture
inoculated
before
the
agar
was
solidified.
Stab
culture
in
agar
was
also
used
for
comparison.
Bacterial
growth
was
quanti-
tatively
evaluated
by
measuring
absorbancy.
Oral
indige-
nous
bacteria
were
used;
enterobacteria
and
other
strains
were
used
for
comparison.
GAM
broth
(Nissui
Co.,
Tokyo,
Japan),
a
medium
for
the
growth
of
anaerobes,
and
GAM
semisolid
medium,
GAM
broth
containing
1.5
g
of
agar
per
liter
of
distilled
water,
were
used
for
the
cultivation
of
both
aerobic
and
anaerobic
strains.
The
composition
of
GAM
broth
was
the
same
as
that
described
in
our
previous
paper
(2).
For
stab
cultures,
seed
cultures
were
stabbed
into
3
ml
of
GAM
semisolid
medium
in
screw-cap
tubes
(12
mm
in
diameter)
by
using
platinum
needles
and
incubated
for
1
to
2
days
at
37°C.
The
extent
of
the
growth
was
determined
by
eye.
For
the
agar-mixed
cultures,
seed
cultures
grown
in
GAM
broth
were
diluted
with
the
same
medium
10-
to
100-fold.
A
0.5-ml
portion
of
each
dilution
was
mixed
in
culture
tubes
with
4.5
ml
of
GAM
semisolid
medium
which
was
kept
molten
at
50°C
in
a
water
bath.
The
mixtures
were
quickly
cooled
and
incubated
in
air
or
in
a
jar
(Tomy
JK-1,
Tominaga
Co.,
Inc.,
Tokyo,
Japan)
filled
with
pure
oxygen
gas
for
1
to
2
days
at
37°C.
The
distance
from
the
medium
surface
to
the
growth
region
was
measured
by
determining
the
absorbancy.
The
absorbancy
was
plotted
against
the
distance.
For
the
shaken
culture,
seed
cultures
(0.05
ml)
obtained
after
24
h
of
incubation
were
inoculated
into
5
ml
of
GAM
broth
in
L-form
culture
tubes
(Monod-type,
18
mm
in
diameter;
Muto
Co.,
Inc.,
Sapporo,
Japan).
The
tubes
were
aerobically
or
anaerobically
shaken
*
Corresponding
author.
at
400
rpm
at
37°C
for
24
h
by
using
a
Gyrotory
incubator
shaker
(New
Brunswick
Scientific
Co.,
Inc.,
Edison,
N.J.).
The
anaerobically
shaken
tubes
were
sealed
after
dissolved
oxygen
in
the
broth
was
evacuated.
To
determine
the
effects
of
shaking,
cultures
in
GAM
broth
were
also
incubated
in
parallel
without
shaking.
The
absorbancy
of
the
liquid
me-
dium
was
measured
by
using
a
Leitz
model
M
photometer
with
an
A
filter.
Observations
were
repeated
three
to
nine
times.
The
relative
bacterial
growth
ratio
(RBGR)
is
the
absorbancy
of
the
aerobically
shaken
culture
divided
by
the
absorbancy
of
the
anaerobically
shaken
culture.
RBGR
values
obtained
for
the
strains
studied
are
given
in
Table
1.
The
results
of
the
stab
cultures
are
also
shown.
The
RBGR
value
is
the
mean
value
of
three
to
nine
determina-
tions,
and
the
values
varied
from
oo
with
obligate
aerobes
to
0
with
obligate
anaerobes.
The
RBGR
of
facultative
anaerobes
was
in
the
range
of
6.0
to
1.8,
and
for
aerotolerant
anaerobes,
the
range
was
from
1.6
to
0.2.
The
effect
of
aeration
on
bacterial
growth
can
be
seen
by
comparing
aerobically
shaken
cultures
with
anaerobically
shaken
cul-
tures.
No
growth
was
observed
for
obligate
anaerobes
in
the
presence
of
air.
In
aerotolerant
anaerobes
(especially
with
Streptococcus
mutans),
the
growth
rate
was
markedly
re-
duced
by
aeration
in
strains
that
showed
RBGR
values
below
0.3,
whereas
the
rate
was
not
affected
in
strains
with
RBGR
values
around
1.0;
furthermore,
growth
was
rather
stimulated
in
most
strains
that
showed
RBGR
values
above
1.3.
In
contrast
to
the
aerobically
shaken
cultures,
little
difference
in
growth
rate
was
observed
between
unshaken
and
anaerobically
shaken
cultures
for
most
aerobes
and
anaerobes.
RBGR
values
thus
obtained
present
a
clearer
classification
than
the
usual
qualitative
growth
indications
obtained
by
using
stab
cultures.
This
method
also
yields
a
quantitative
estimation
for
the
classification
of
Brock
(1).
Results
with
the
agar-mixed
culture
method
are
shown
in
Fig.
1.
The
growth
characteristics
of
obligate
aerobes,
fac-
ultative
anaerobes,
aerotolerant
anaerobes,
and
obligate
anaerobes
could
be
clearly
visualized
by
this
method.
Growth-inhibited
zones
with
the
agar-mixed
culture
were
clearly
observed
below
the
medium
surface
for
aerotolerant
anaerobes
and
obligate
anaerobes.
The
depth
was
less
in
air
than
in
pure
oxygen,
and
it
was
also
less
in
cultures
with
an
inoculation
of
107
CFU
than
in
cultures
inoculated
with
104
CFU.
The
oxidation-reduction
potential
in
the
medium
de-
971
APPL.
ENVIRON.
MICROBIOL.
972
NOTES
TABLE
1.
Bacterial
growth
in
stab
and
shaken
cultures
Growth
in
broth
(A630)
Growth
on
semisolid
agar
Organism
type
and
strain'
Not
Shaken
Anaerobically
RBGRb
d
shaken
in
air
shaken
Surface'
Depth
Obligate
aerobes
Micrococcus
luteus
NCTC
2665
0.02
0.15
0.00
+
+
Neisseria
mucosa
S-li0
0.05
2.00
0.04
50.0
±
1.4
++
Pseudomonas
aeruginosa
S-2
0.09
0.07
0.01
7.0
±
0.1
++
Facultative
anaerobes
Escherichia
coli
IID
861
0.58
3.05
0.51
6.0
±
1.2
++
+
Escherichia
coli
K-67
0.52
2.75
0.49
5.6
±
2.0
++
+
Escherichia
coli
B/r
0.46
1.53
0.52
2.9
±
0.1
++
+
Proteus
vulgaris
IID
874
0.36
0.93
0.42
2.2
±
0.0
++
+
Staphylococcus
aureus
IID
671
0.44
0.95
0.48
2.0
±
0.1
++
+
Salmonella
enteritidis
IID
604
0.61
1.08
0.61
1.8
±
0.2
++
+
Aerotolerant
anaerobes
Streptococcusfaecalis
ATCC
9756
0.83
1.32
0.82
1.6
±
0.8
+
+
Streptococcus
mutans
MT
703
0.23
0.18
0.13
1.4
±
0.2
+
+
Streptococcus
mutans
Pk
1
0.59
1.42
0.98
1.4
±
0.3
+
+
Streptococcus
faecalis
ATCC
19433
0.96
1.42
1.08
1.3
±
0.6
+
+
Streptococcus
pyogenes
IID
689
0.54
0.35
0.28
1.3
±
0.4
+
+
Streptococcus
mutans
OMZ
175
0.41
0.54
0.47
1.1
±
0.5
+
+
Streptococcus
mutans
B
13
0.42
0.52
0.46
1.1
±
0.5
+
+
Streptococcus
sanguis
S-9
0.25
0.24
0.24
1.0
±
0.4
+
+
Streptococcus
mutans
Ingbritt
0.14
0.15
0.16
1.0
±
0.2
+
+
Streptococcus
mutans
1089
0.15
0.14
0.18
0.8
±
0.5
+
+
Streptococcus
mutans
BHT
0.35
0.14
0.33
0.4
±
0.0
+
+
Streptococcus
salivarius
S-6
0.54
0.13
0.52
0.3
±
0.0
+
+
Lactobacillus
acidophilus
ATCC
0.20
0.06
0.18
0.3
±
0.1
+
+
4356
Streptococcus
sanguis
ATCC
10557
0.39
0.13
0.38
0.3
±
0.1
+
+
Streptococcus
mitior
(mitis)
S-8
0.21
0.05
0.25
0.2
±
0.1
+
+
Obligate
anaerobes
Veillonella
alcalescens
ATCC
17748
0.10
0.02
0.12
0.2
±
0.1
-
+
Propionibacterium
acnes
ATCC
0.97
0.01
0.99
0.0
±
0.1
-
+
11827
Fusobacterium
nucleatum
IID
891
0.43
0.01
0.37
0.0
±
0.1
-
+
a
Strains
of
S
series
were
isolated
by
our
laboratory.
b
Mean
of
three
to
nine
determinations
±
the
standard
deviation.
C
The
extent
of
growth
was
examined
by
eye.
+
+,
Growth
at
the
whole
surface
area;
+,
growth
in
a
small
region
around
the
stabbed
point;
-,
no
growth.
d
+,
Growth
at
>5
mm
below
the
surface
region;
-,
no
growth.
termined
by
resazurin
changed
with
Eh
+0.051
V
in
the
zone
where
the
growth
was
inhibited.
The
result
did
not
com-
pletely
agree
with
the
order
of
aerotolerance
obtained
by
RBGR
values.
As
the
depth
varied
with
the
inoculated
concentration
of
organisms,
the
growth
in
agar-mixed
cul-
tures
may
involve
complicated
effects
of
oxygen
consump-
tion
and
accumulation
of
metabolites.
As
described
above,
a
quantitative
index
was
obtained
that
is
useful
for
the
classification
of
microorganisms
when
they
are
cultured
under
well-defined
conditions
and
their
growth
is
measured
quantitatively.
The
shaken
culture
with
a
liquid
medium
is
suitable
for
making
culture
conditions
as
constant
as
possible.
In
conclusion,
RBGR
is
a
simple
means
of
giving
a
quantitative
indication
of
oxygen
relation.
The
plot
of
absor-
bancy
versus
depth
may
also
be
useful.
Both
RBGR
and
NOTES
973
Pseudomonas
aeruginosa
S-2
B
:~~~~~~~...I......!...
..............
.............I...
0
mm
-10
mm
-20
mm
-30
mm
0
mm
-10
mm
-20
mm
-30
mm
C
i
s4
:
: :
i
C
Absorbanc
y
10
Stetccu
:ua.
iI*g.rTtt
D
Fusobacterium
nucleatum
IID
891
FIG.
1.
Growth
characteristics
and
absorbancy
patterns
of
organisms
in
agar-mixed
cultures.
The
distance
from
the
medium
surface
to
the
growth
region
was
determined
by
measuring
the
absorbancy,
and
the
absorbancy
was
plotted
against
the
distance.
Organisms
shown
are
representative
of
obligate
aerobes
(A),
facultative
anaerobes
(B),
aerotolerant
anaerobes
(C),
and
obligate
anaerobes
(D).
absorbancy
plot
may
lend
themselves
to
automated
systems
analysis.
The
authors
thank
K.
Oikawa, H.
Tani,
and
0.
Nishikaze
of
the
School
of
Dentistry,
Hokkaido
University,
for
kindly
giving
direc-
tions
and
encouragement.
LITERATURE
CITED
1.
Brock,
T.
D.
1974.
Biology
of
microorganisms,
p.
313-318.
Prentice-Hall,
Inc.,
Englewood
Cliffs,
N.J.
2.
Kikuchi,
H.
E.,
and
T.
Suzuki,
1984.
An
electrophoretic
analysis
of
superoxide
dismutase
in
Campylobacter
spp.
J.
Gen.
Micro-
biol.
130:2791-2796.
VOL.
52,
1986
A
5
A
b
so
rba
ncy
10
0
mm
-10
mm
-20
mm
-
30
mm
0
mm
-10..
-20
mm
-30
mm
Escherichia
coli
K12
