APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
Feb.
1976,
p.
268-273
Copyright
©
1976
American
Society
for
Microbiology
Vol.
31,
No.
2
Printed
in
U.S.A.
Characteristics
of
Bacteria
Isolated
by
the
Anaerobic
Roll-
Tube
Method
from
Cheeses
and
Ground
Beef
1
W.
M.
GRAY
AND
M.
G.
JOHNSON*
Departments
of
Food
Science
and
Microbiology,
Clemson
University,
Clemson,
South
Carolina
29631
Received
for
publication
11
September
1975
In
this
study
the
methods
of
Hungate
were
used
to
quantitate
the
anaerobic
bacteria
present
in
commercially
available
ground
beef,
cheddar
cheese,
and
German
hand
cheese.
Of
235
anaerobic
roll-tube
isolates
from
ground
beef
and
German
hand
cheese,
all
were
facultative
anaerobes.
Of
213
anaerobic
roll-tube
isolates
from
cheddar
cheese,
91%
were
facultative
anaerobes
and
9%
were
obligate
anaerobes.
Using
results
of
biochemical
tests,
14
of
the
17
obligately
anaerobic
isolates
from
cheddar
cheese
were
Propionibacterium
acnes,
two
were
strains
of
Propionibacterium
that
could
not
be
speciated,
and
one
was
tenta-
tively
identified
as
a
strain
of
Streptococcus
evolutus.
Obligate
anaerobes
were
estimated
to
be
present
in
the
cheddar
cheese
at
a
level
of
about
106/g.
The
possible
significance
of
these
levels
of
P.
acnes
in
nonsterile
foods
is
discussed.
Several
of
the
recommended
methods
in
ma-
jor
reviews
concerning
the
isolation
and
enu-
meration
of
anaerobes
in
foods
(6,
8,
14)
allowed
some
exposure
of
microbes
to
oxygen.
Even
the
methods
Claybaugh
used
in
searching
for
obli-
gate
anaerobes
in
dairy
products
did
not
ex-
clude
oxygen
(4).
Also,
methods
recommended
for
the
examination
of
canned
foods
by
the
Food
and
Drug
Administration
(19)
involve
exposing
food
samples
to
some
oxygen.
All
these
methods
are
generally
satisfactory
for
the
recovery
of
vegetative
cells
and
spores
of
aerotolerant
anaerobes
and
are
usually,
but
not
always,
adequate
for
enumeration
of
clostridia
in
foods.
However,
methods
that
allow
some
exposure
to
oxygen
would
not
be
adequate
for
the
recovery
of
strictly
anaerobic
bacteria
from
foods
because
these
organisms
will
die
if
ex-
posed
to
oxygen
(10).
By
definition
then,
anaer-
obic
methods,
such
as
the
Hungate
roll-tube
technique
(10),
are
necessary
to
adequately
quantitate
the
obligately
anaerobic
organisms
in
the
food,
including
those
organisms
that
may
be
injured
or
debilitated
(15).
Thus,
in
this
study,
roll-tube
techniques
and
habitat-simu-
lating
media
were
used
to
isolate
and
charac-
terize
anaerobic
bacteria
present
in
three
foods:
ground
beef,
cheddar
cheese,
and
German
hand
cheese.
A
preliminary
report
of
this
work
was
pre-
sented
earlier
(W.
M.
Gray
and
M.
G.
Johnson,
Abstr.
Annu.
Meet.
Am.
Soc.
Microbiol.
1974,
E104,
p.
18).
'
Technical
contribution
no.
1297
of
the
South
Carolina
Agricultural
Experiment
Station,
Clemson
University,
Clemson,
S.C.
29631.
(This
paper
was
taken
in
part
from
a
thesis
submitted
by
W.
M.
G.
to
Clemson
University,
Clemson,
S.C.,
in
partial
fulfillment
of
the
re-
quirements
for
the
M.S.
degree
[1974].)
MATERIALS
AND
METHODS
Food
sample
collection
and
storage.
All
foods
were
obtained
from
local
retail
outlets.
Refrigerated
ground
beef
was
purchased
over
a
3-month
period
in
454-g
plastic
tube
packages.
Refrigerated
cheddar
cheese
was
purchased
in
1.6-kg
wedges,
which
were
freshly
cut
from
large
cheese
wheels
and
wrapped
in
Saran
film.
Frozen
German
hand
cheese
was
pur-
chased
in
175-g
foil-wrapped
packages.
All
samples
were
refrigerated
at
7
C
and
sampled
after
1
or
2
days
and
1
month.
Food
sample
preparation.
Core
samples
of the
foods
were
aseptically
transferred
and
blended
in
a
500-ml
Waring
blender
jar
containing
enough
prere-
duced
anaerobically
sterilized
(PRAS)
medium
to
yield
a
10-fold
dilution.
The
PRAS
dilution
medium
was
of
the
same
composition
as
that
used
for
culture
of
the
specimen,
except
that
it
contained
no
agar.
The
apparatus
with
core
sampler
inserted
was
con-
tinuously
flushed
with
oxygen-free
CO2
(Fig.
1).
Media
composition
and
preparation.
The
PRAS
media
were
prepared
using
methods
described
by
Hungate
(10)
and
were
dispensed
into
25-
by
142-mm
roll
tubes
for
enumeration
studies
and
into
16-
by
150-mm
tubes
for
stock
culture
maintenance.
Tubes
were
sealed
with
butyl
rubber
stoppers.
Screw-cap
roll
tubes
(18
by
142
mm),
sealed
with
butyl
rubber
septa
(1,
12)
were
used
for
fermentation
studies.
All
tubes
were
obtained
from
Bellco
Glass,
Inc.,
Vine-
land,
N.J.
The
basal
medium
(designated
PYG)
consisted
of
1%
(wt/vol)
each
of
peptone
(Difco
Laboratories,
De-
troit,
Mich.),
yeast
extract
(Difco),
and
glucose,
2%
(vol/vol)
each
of
inorganic
salt
solutions
A
and
B
268
OBLIGATE
ANAEROBES
FROM
CHEESE
EXTRUSION
ROD
ALUMINUM
SAMPLER
FOOD
SAMPLE
WAR
NG
BLENDER
JAR
DILUENT
(PRASI
FIG.
1.
Apparatus
for
anaerobically
sampling
and
blending
food.
Cores
(approximately
2-cm
ID)
of
food
were
cut
with
the
sampler
and
extruded
with
a
stainless-steel
rod
into
a
500-ml
blender
jar.
Anaero-
biosis
was
maintained
in
the
jar
during
blending
by
sparging
the
diluent
with
oxygen-free
CO2
gas
via
a
14-gauge
stainless-steel
transfer
needle.
(10),
0.0001%
(vol/vol)
resazurin,
0.05%
(wt/vol)
L-
cysteine-HClH2O,
and
0.85%
(wt/vol)
sodium
bicar-
bonate.
After
sterilization
under
a
100%
CO2
gas
phase
this
medium
had
a
pH
of
6.7.
When
proteose
peptone
no.
3
(Difco)
was
substituted
for
peptone,
the
resulting
medium
(PPYG)
had
an
identical
ini-
tial
pH.
A
habitat-simulating
medium
was
prepared
by
adding
a
cheese
extract
to
PYG
to
give
a
final
me-
dium
concentration
of
1%
(vol/vol)
and
was
desig-
nated
PYG-CE.
This
PYG-CE
medium
had
a
pH
of
6.8
under
100%
CO2.
Cheese
extract
was
prepared
by
blending
a
100-g
cheese
sample
with
500
ml
of
warm
2%
(wt/vol)
sodium
citrate
solution,
boiling
mildly
and
filtering
this
mixture,
centrifuging
the
filtrate
at
4
C,
and
discarding
the
fat
layer.
Another
modi-
fied
PYG
medium
was
prepared
by
adding
cyclohex-
imide
(grade
B;
Calbiochem,
Los
Angeles,
Calif.)
to
give
a
final
concentration
of
100
,ug/ml.
The
result-
ing
medium
had
a
pH
of
6.9
under
100%
CO2.
Plate
count
medium
(Difco)
was
modified
(MPC)
by
adding
0.85%
(wt/vol)
sodium
bicarbonate,
0.05%
(wt/vol)
L-
cysteine-HClH2O,
and
0.0001%
(vol/vol)
resazurin.
The
pH
of
this
medium
was
6.9
under
100%
CO2.
Agar,
when
used,
was
present
at
2%
(wt/vol).
For
isolation
of
aerobic
bacteria
from
foods,
media
of
the
same
composition
as
those
used
for
isolation
of
anaerobes
were
employed,
except
that
tubes
of
me-
dia
were
sterilized
aerobically
with
no
CO2
gas
phase
and
sodium
bicarbonate
and
L-cysteine-
HCl-H20
were
deleted
except
in
some
tests
noted
below.
Medium
inoculation
and
incubation.
Triplicate,
tempered-agar
medium
roll
tubes
were
anaerobi-
cally
inoculated
with
anaerobic
dilutions
of
the
food
sample
and
solidified.
Aerobic
spread
plates
were
also
prepared
in
triplicate
from
the
anaerobic
dilu-
tion
series.
Roll
tubes
were
incubated
14
days
at
30
C,
or
in
some
cases
at
37
C,
before
counting.
Aerobic
plates
were
incubated
at
the
same
tempera-
tures
for
5
to
7
days
when
colony
counts
were
maxi-
mal.
Estimation
of
facultative
anaerobe
population.
Aerobic
spread
plates
with
70
to
80
colonies
or
less
were
replica
plated
by
the
Lederberg
method
(11)
onto
plates
of
the
same
medium
used
for
isolation.
Transferred
colonies
capable
of
growing
anaerobi-
cally
were
counted
on
these
plates
after
incubation
for
5
to
7
days
in
a
GasPak
anaerobic
jar
(Baltimore
Biological
Laboratory,
Cockeysville,
Md.)
equipped
with
a
disposable
H2/CO2
generator,
palladium
cata-
lyst,
and
a
methylene
blue
strip
as
an
indicator
of
anaerobiosis.
Selection
of
anaerobic
isolates.
Well-isolated
col-
onies
from
higher
dilution
roll
tubes
(10-s
to
10-8)
were
picked
anaerobically
(under
CO2)
with
a
stain-
less-steel
wire
loop
into
the
PRAS
broth
form
of
the
isolation
medium
in
16-
by
150-mm
tubes.
These
tubes
were
continuously
flushed
with
CO2,
closed
with
recessed
butyl
rubber
stoppers
and
incubated
at
30
C
for
48
to
72
h.
A
0.1-ml
portion
of
anaerobic
broth
stock
culture
of
each
isolate,
obtained
by
roll-
tube
methods
(9,
10),
was
transferred
to
the
aerobic
form
of
the
isolation
medium
using
a
sterile
syringe
(12).
Isolates
that
grew
under
these
aerobic
condi-
tions
were
eliminated
from
consideration
as
obligate
anaerobes.
Inability
of
anaerobic
isolates
to
grow
aerobically
was
also
confirmed
by
spread
plating
0.2-ml
amounts
of
each
anaerobic
stock
culture
on
aerobic
plates
and
incubating
these
for
2
weeks
at
30
C.
Isolates
that
formed
no
visible
colonies
on
these
aerobic
spread
plates
and
grew
only
in
anaerobic
agar
roll
rubes
were
considered
obligate
anaerobes.
Obligately
anaerobic
isolates
were
subsequently
purified
by
culturing
serial
dilutions
in
agar
roll
tubes
and
picking
well-isolated
colonies
(10).
Characterization
of
obligately
anaerobic
iso-
lates.
The
Gram-staining
reaction
of
all
purified
anaerobic
isolates
was
observed
by
staining
samples
from
young
PYG
broth
cultures
using
smears
of
Escherichia
coli
9002
and
Staphylococcus
aureus
S-6
as
controls.
Cell
size
was
determined
from
photomi-
crographs
of
wet
mounts
of
48-
to
72-h
cultures
from
PRAS
PYG.
The
peptone-yeast
extract
(PY)
broth
and
carbo-
hydrate
concentrations
used
in
fermentation
tests
were
those
described
in
the
Anaerobe
Laboratory
Manual
(9).
Hungate
screw-cap
tubes
with
butyl
rubber
septa
were
used
for
all
test
media
(12).
The
sterility
of
the
rubber
septa
in
these
tubes
was
maintained
by
capping
the
sealed
tubes
with
stain-
less-steel
18-mm
closures.
All
transfers
were
per-
formed
via
syringes
with
25-gauge
needles
inserted
through
the
septa
(12).
Five
drops
of
a
young
culture
of
each
anaerobic
isolate
grown
in
PY
broth
were
used
to
inoculate
the
various
carbohydrate
test
me-
dia.
All
tests
were
performed
in
duplicate.
Culture
pH
values
were
determined
in
the
culture
tubes
using
a
Corning
model
12
research
pH
meter
and
a
Corning
semimicro
combination
pH
electrode
(no.
476050).
The
difference
in
the
pH
produced
by
the
isolate
grown
in
PY
(generally
7.0
+
0.2)
and
the
pH
produced
in
PY
broth
with
an
added
carbohydrate
was
taken
as
an
index
of
the
ability
of
the
organism
VOL.
31,
1976
269
270
GRAY
AND
JOHNSON
to
ferment
that
carbohydrate.
All
other
tests
re-
ported
below
were
performed
as
described
in
the
Anaerobe
Laboratory
Manual
(9),
except
that
an
incubation
temperature
of
30
C
was
used
in
all
cases.
RESULTS
Ground
beef.
The
numbers
and
percentages
of
isolates
from
the
three
foods
examined
that
were
able
to
grow
as
aerobes,
facultative
anae-
robes,
or
obligate
anaerobes
are
summarized
in
Table
1.
Of
182
ground
beef
isolates
examined
from
anaerobic
rool
tubes,
all
were
facultative
anerobes.
No
obligate
anaerobes
were
detected
using
the
PRAS
forms
of
PYG,
PPYG,
MPC,
nutrient
agar,
or
PPYG
with
hamburger
ex-
tract
and
10%
bovine
rumen
fluid
and
gas
phases
of
100%
CO2,
97%
CO2/3%
H,
or
80%
N,l
10%
H2/10%
CO.
Cheddar
and
German
hand
cheese.
Prelimi-
nary
results
of
culturable
cell
counts
for
ched-
dar
cheese
suggested,
erroneously,
that
a
major
fraction
of
the
microflora
consisted
of
obligately
anaerobic
organisms.
However,
when
cysteine-
hydrochloride
and
sodium
bicarbonate
were
de-
leted
from
the
aerobic
plating
media
the
appar-
ent
difference
in
anaerobic
and
aerobic
counts
disappeared.
Subsequent
experiments
showed
that
the
pH
of
9.2
of
the
aerobic
medium
with
sodium
bicarbonate
present
was
responsible
for
the
lower
aerobic
counts
observed.
Presence
of
cysteine
without
sodium
bicarbonate
did
not
affect
the
aerobic
counts.
In
further
experi-
ments,
cysteine
and
sodium
bicarbonate
were
deleted
from
the
aerobic
media.
Cultures
initially
isolated
by
anaerobic
means
were
tested
for
ability
to
grow
aerobi-
cally,
and
91%
(194/213)
of
the
cheddar
cheese
isolates
and
100%
(53/53)
of
the
German
hand
cheese
isolates
were
able
to
do
so
(Table
1).
Few
of
the
organisms
present
in
cheddar
cheese
were
sporeformers,
since
counts
of
heated
sam-
ples
were
only
100
to
1,000/g.
Only
two
isolates
tentatively
identified
as
obligate
anaerobes
were
heat
tolerant.
Whether
these
were
spore-
formers
could
not
be
confirmed
before
they
were
lost
in
subculturing.
Most
importantly,
a
total
of
17
obligately
anaerobic
isolates
from
cheddar
cheese
were
recovered
and
purified.
As
for
ground
beef,
no
one
medium,
gas
phase,
or
incubation
temperature
(30
or
37
C)
was
ob-
viously
superior
for
isolation
of
these
orga-
nisms. Since
the
majority
of
the
isolates
(15/17)
were
picked
from
10"
or
10-7
dilution
roll
tubes,
these
isolates
were
present
at
a
level
of
at
least
106/g
in
samples
of
this
cheese
exam-
ined.
Replica
plating
of
organisms
isolated
aerobi-
cally
onto
aerobic
media
followed
by
anaerobic
incubation
showed
that
95%
(208/220)
of
the
cheddar
cheese
isolates
were
capable
of
faculta-
tive
anaerobic
growth.
Of
the
aerobic
isolates
from
German
hand
cheese,
79%
(94/119)
were
capable
of
facultative
anaerobic
growth.
When
the
same
isolates
were
replicated
onto
aerobic
media
and
then
incubated
aerobically,
92%
(339/435)
formed
colonies.
Obligately
anaerobic
isolates.
All
17
isolates
were
gram
positive,
although
several
orga-
nisms
stained
unevenly.
Sixteen
of
the
strains
were
rod
shaped
and
coryneform
in
their
mor-
phology,
with
mean
sizes
ranging
from
0.6
to
1.0
,um
in
length.
Of
these,
isolate
number
91
was
particularly
pleomorphic,
ranging
from
a
rod
to
a
coccal
shape.
The
remaining
isolate,
number
69,
was
a
coccal-shaped
bacterium
with
a
mean
diameter
of
1.0
,am
and
occurred
singly
or
in
pairs.
All
isolates,
except
numbers
69
and
124,
re-
mained
obligately
anaerobic
on
subculture.
These
two
were
obligate
anaerobes
upon
initial
isolation
and
for
several
weeks
thereafter,
but
after
four
to
five
transfers
they
were
able
to
grow
aerobically
in
PYG
broth
or
on
PYG
agar
plates.
The
ability
of
the
anaerobic
isolates
to
pro-
TABLE
1.
Ability
of
isolates
obtained
from
foods
by
anaerobic
or
aerobic
methods
to
grow
as
obligate
anaerobes,
facultative
anaerobes,
or
aerobes
No.
of
iso-
Isolates
(%)
that
were:
Food
Isolation
Culturable
count/g
lates
exam-
method
(range)
ined
Obligate
Facultative
Obligate
aerobes
anaerobes
anaerobes
Ground
beef
Aerobic
6.5
x
104-1.4
x
10W
1,030
50 50
0
Anaerobic
1.7
x
10:$-3.8
x
10o
182
0
100
0
Cheddar
cheese
Aerobic
Anaerobic
7.9
x
10'i-2.5
x
10f
220
5
95
0
6.3
x
106-1.3
x
10?
213
0 91
9
German
hand
cheese
Aerobic
Anaerobic
1.6
x
107-2.0
x
107
119
21
79
0
1.6
x
10X-2.5
x
107
53
0
100
0
APPL.
ENVIRON.
MICROBIOL.
OBLIGATE
ANAEROBES
FROM
CHEESE
duce
indole
from
tryptophan,
reduce
nitrate
to
nitrite,
and
ferment
various
carbohydrates
is
summarized
in
Table
2.
No
isolate
digested
meat,
and
only
isolate
69
liquified
gelatin.
All
isolates
produced
an
acid
clot
in
milk
except
isolate
91,
which
caused
no
change
in
milk.
Carbohydrate
tests
(Table
2)
showed
that
all
cultures
fermented
glucose,
fructose,
and
man-
nose.
Isolates
69
and
124
also
fermented
sucrose
and
trehalose.
Isolate
69
was
the
only
one
able
to
ferment
galactose.
Production
of
volatile
and
nonvolatile
fatty
acids
and
alcohols
was
determined
by
gas
chro-
matographic
analyses
of
solvent
extracts
of
acidified
broth
culture
samples
from
eight
se-
lected
obligately
anaerobic
isolates
grown
in
PRAS
PYG
medium
(Table
3).
Isolates
58,
75,
92,
and
122
produced
similar
amounts
of
acetate
(0.1
to
0.2
meq/100
ml),
propionate
(0.8
to
0.95
meq/100
ml),
and
lactate
(0.18
to
0.24
meq/100
ml),
and
trace
amounts
of
pyruvate
(0.03
to
0.12
meq/100
ml)
and
succinate
(0.03
to
0.09
meq/100
ml).
By
comparison,
isolates
121
and
160
pro-
duced
less
propionate
(0.5
meq/100
ml)
and
es-
sentially
no
acetate,
but
small
amounts
of
lac-
tate,
pyruvate,
and
succinate.
Isolate
91
pro-
duced
considerably
more
acetate,
propionate,
and
succinate
than
the
other
isolates.
Isolate
69
produced
primarily
lactate
and
a
small
amount
of
acetate.
TABLE
2.
Some
biochemical
characteristics
of
obligately
anaerobic
isolates
from
cheddar
cheese
Carbohydrate
and
Isolate
class"
Isolate
no.
other
tests
II
III
47
57
69
91
124
160
Cellobiose
_
b
_
-
-
-
-
-
-
DL-Erythritol
-
-
-
-
a
-
-
-
-
Esculin
D-Fructose
a'
a
wI)
a
a
a
a
a
w
D-Galactose
-
-
-
-
-
a
Glucose
a
a
w
a
a
a
a
a
w
Lactose
-
-
-
-
-
a
D-(+)-Maltose
-
-
-
-
-
a
D-Mannitol
D-(+)-MannOse
a
w
w
w
a
a
a
a
w
Melibiose
-
- -
-
-
D-Sorbitol
-
-
-
-
-
Starch
-
-
-
-
-
Sucrose
-
-
-
-
-
a
-
a
Trehalose
-
-
-
-
-
a
-
a
D-Xylose
-
-
-
-
-
-
-
Esculin
hydrolysis
NHW
NH
NH
NH
NH
NH
NH
NH
NH
Starch
hydrolysis
NHc
NH
NH
NH
NH
NH
NH
NH
NH
Indole
production
+
+
+
-
+
-
-
+
Nitrate
reduction
+
+
+
+
+
-
-
+
+
Class
I
includes
isolates
number
120, 122,
123, 139,
and
161;
class
II
includes
isolates
number
58,
92,
143,
and
164;
class
III
includes
isolates
number
75
and
121.
-,
No
decrease
or
decrease
of
less
than
0.5
pH
units;
w,
decrease
of
0.5
to
0.9
pH
units;
a,
decrease
of
1.0
or
more
pH
units.
'
NH,
No
hydrolysis.
TABLE
3.
Fermentation
patterns
produced
by
selected
obligately
anaerobic
isolates
in
PYG
medium"
Isolate
class
Isolate
no.
Acetic
Propionic
Lactic
Succinic
I
122
20"
95
21
9
II
58
10
80
24
3
II
92
20
95
18
6
III
75
10
80
17
0
III
121
0
55
27
1
69
20
0
130
4
91
140
250
0
28
160
5
50
18
0
"Samples
were
prepared
and
analyzed
by
gas
chromatography
according
to
the
methods
described
in
the
Anaerobe
Laboratory
Manual
(9).
b
Values
are
100
(number
of
milliequivalents/100
ml
of
culture
broth).
VOL.
31,
1976
271
272
GRAY
AND
JOHNSON
DISCUSSION
The
obligately
anaerobic
bacteria
examined
were
only
the
most
numerous
ones
in
the
ched-
dar
cheese,
since
they
were
from
colonies
picked
from
the
highest
dilution
roll
tubes.
Thus,
it
is
possible
there
were
other
anaerobic
bacteria
present
as
a
smaller
fraction
of
the
total
food
microflora
that
could
not
be
isolated
by
the
procedure
used
here.
This
fraction
in
hamburger
and
German
hand
cheese
perhaps
was
too
small
to
permit
isolation
of
any
anaer-
obes.
A
second
possibility
is
that
few
or
no
anaerobes
were
present
in
these
two
foods.
Isolate
69,
the
only
coccal-shaped
anaerobe
found,
is
gram
positive
and
similar
in
size
and
morphology
to
some
streptococci.
The
carbohy-
drates
fermented,
biochemical
activities,
and
compounds
produced
from
glucose
are
quite
similar
to
those
listed
for
Peptostreptococcus
evolutus
in
the
7th
edition
of
Bergey's
Manual
(2).
The
tendency
of
our
isolate
to
become
aero-
tolerant
on
subculturing
also
agrees
with
that
description.
Prevot
(16)
described
a
similar
or-
ganism,
Streptococcus
evolutus,
as
occurring
widely
in
the
respiratory
and
digestive
tracts
of
man.
In
the
8th
edition
of
Bergey's
Manual
(18,
p.
523),
Peptostreptococcus
evolutus
is
no
longer
recognized
as
a
valid
species
of
the
genus
Pep-
tostreptococcus.
Because
it
grows
fairly
well
in
air
after
several
passages
and
produces
primar-
ily
lactic
acid
from
glucose,
it
was
recom-
mended
(18)
that
this
organism
be
classified
with
Streptococcus.
Unfortunately,
the
authors
of
the
section
on
streptococci
did
not
recognize
this
organism.
Fourteen
of
the
other
16
obligately
anaerobic
isolates
exhibited
coryneform
morphology
and
generally
had
the
same
size,
Gram
reaction,
and
biochemical
test
reactions
as
those
de-
scribed
for
the
organism
Propionibacterium
acnes
(3,
5,
9,
13).
Of
the
carbohydrates
tested
only
the
negative
reaction
for
galactose
disa-
grees
with
the
typical
fermentation
pattern
found
for
72
strains
of
P.
acnes
by
Pulverer
and
Ko
(17).
However,
it
was
reported
(9)
that
galactose
was
not
always
fermented
by
strains
of
P.
acnes.
All
our
other
test
results
for
these
14
isolates
are
in
general
agreement
with
those
described
by
these
workers
and
those
described
for
324
strains
of
P.
acnes
in
the
8th
edition
of
Bergey's
Manual
(13).
It
was
not
possible
to
classify
the
other
two
isolates,
91
and
124,
further
than
to
the
genus
level,
Propionibacterium.
Isolate
91
had
nega-
tive
indole
and
nitrate
tests
and
produced
large
amounts
of
propionic
acid.
Had
isolate
91
also
fermented
sucrose
and
maltose,
which
it
did
not,
one
might
tentatively
identify
this
isolate
as
Propionibacterium
granulosum.
Since
completing
this
work,
it
has
come
to
our
attention
that
anaerobic
bacteria,
such
as
Propionibacterium
species,
may
grow
better
and
more
actively
ferment
sugars
such
as
ga-
lactose,
sucrose,
and
maltose
if
Tween
80
at
a
final
concentration
of
0.02%
(wt/vol)
is
added
to
the
test
broth
(Anaerobe
Newsletter
no.
2,
Oc-
tober
1974,
p.
3-4,
V.P.I.
Anaerobe
Laboratory,
Blacksburg,
Va.).
The
absence
of
this
surfac-
tant
in
our
test
media
and
the
media
of
others
(9,
17)
may
account
for
the
variability
we
and
others
have
observed
in
the
fermentation
of
these
three
sugars
by
Propionibacterium
spe-
cies.
Starter
cultures
composed
of
species
of
strep-
tococci
and
lactobacilli
were
probably
used
in
the
production
of
the
cheddar
cheese
examined
in
this
study.
These
starter
cultures
undoubt-
edly
produced
lactate
as
a
major
fermentation
product.
P.
acnes
can
ferment
lactate
to
propionate
and
acetate
and
is
normally
found
associated
with
the
normal
skin
and
intestinal
tracts
of
mammals
and
poultry
(9,
p.
50;
13).
Because
of
the
materials
they
produce
and
ferment,
lactic
acid
bacteria
and
propionibacteria
can
share
a
commensal
relationship
in
cheese
products
(7).
It
is
therefore
not
too
surprising
that
we
found
a
large
number
of
P.
acnes
(106/g)
in
the
cheddar
cheese
we
examined.
The
propionibacteria
we
detected
in
the
ched-
dar
cheese
likely
became
established
by
one
of
three
mechanisms.
These
bacteria
initially
may
have
been
present
in
fairly
high
numbers
in
raw
milk
and
survived
the
pasteurization
treatment
given
the
milk
prior
to
starter
cul-
ture
inoculation.
A
second
possibility
is
that
these
organisms
were
introduced
by
direct
hu-
man
skin
contact
during
the
salting,
milling,
and
handling
of
the
curd
prior
to
cheese
aging.
A
third
possibility
is
that
there
were
initially
present
only
a
small
number
of
these
bacteria,
and
they
increased
to
the
high
numbers
ob-
served
only
after
cheese
aging
and
curing
when
a
favorable
reduced
oxidation-reduction
poten-
tial
was
created
by
the
growth
of
the
other
microflora
present.
Strains
of
both
P.
acnes
and
S.
evolutus
iso-
lated
from
clinical
specimens
have
been
impli-
cated
in
various
pathological
processes
(9,
16,
17).
However,
these
organisms
when
competing
with
the
mixed
microflora
present
in
the
ched-
dar
cheese
did
not
appear
to
exert
any
patho-
genic
effects
on
one
of
us
(W.M.G.)
who
ate
considerable
amounts
of
this
cheese.
The
desirable
changes
anaerobes
may
exert
APPL.
ENVIRON.
MICROBIOL.
OBLIGATE
ANAEROBES
FROM
CHEESE
273
on
food
flavor
and
texture
during
food
process-
ing
have
been
little
studied,
except
in
the
aging
of
Swiss
cheeses
(7).
Cheeses
and
ground
beef
are
normally
held
at
refrigeration
temperatures
of
0
to
10
C.
It
is
possible
that
there
were
present
in
the
foods
we
examined
psychrophilic
and/or
psychrotrophic
strains
of
obligate
anaerobes
that
could
not
be
cultivated
and
isolated
at
the
incubation
tem-
peratures
of
30
and
37
C
used.
We
are
aware
of
one
other
report
in
which
the
Hungate
roll-tube
method
was
used
in
an
at-
tempt
to
isolate
obligate
anaerobes
from
foods
(D.
P.
Ward,
M.
D.
Pierson,
and
K.
M.
Rice,
Abstr.
Annu.
Meet.
Inst.
Food
Technol.
1975,
p.
149).
They
were
unable
to
isolate
any
obligate
anaerobes
from
either
unpasteurized
or
pas-
teurized
crabmeat
and
reported
that
the
pre-
dominant
bacteria
apparently
were
homofer-
mentative
lactobacilli.
Other
foods
should
be
examined
for
their
ob-
ligate
anaerobe
content
to
determine
what
role,
if
any,
these
organisms
have
in
assessing
food
quality
and
food
safety.
ACKNOWLEDGMENTS
The
assistance
of
W.
E.
C.
Moore,
Virginia
Polytechnic
Institute
and
State
University,
with
gas-liquid
chromato-
graphic
analyses
of
broth
culture
extracts
and
several
help-
ful
discussions
with
M.
J.
B.
Paynter,
Microbiology
Depart-
ment,
Clemson
University,
are
gratefully
acknowledged.
This
work
was
supported
by
a
grant
to
M.
G.
J.
by
the
Clemson
University
Faculty
Basic
Research
Committee
and
by
funds
from
the
U.S.
Department
of
Agriculture
Hatch
Project
SC00006
granted
to
the
South
Carolina
Agri-
cultural
Experiment
Station.
Finally,
this
paper
is
dedicated
to
R.
E.
Hungate,
whose
pioneering
work
made
this
study
possible.
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