APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1976, p. 268-273 Vol. 31, No.

2
Copyright © 1976 American Society for Microbiology 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- (This paper was taken in part from a thesis
jor reviews concerning the isolation and enu- submitted by W. M. G. to Clemson University,
meration of anaerobes in foods (6, 8, 14) allowed Clemson, S.C., in partial fulfillment of the re-
some exposure of microbes to oxygen. Even the quirements for the M.S. degree [1974].)
methods Claybaugh used in searching for obli-
gate anaerobes in dairy products did not ex- MATERIALS AND METHODS
clude oxygen (4). Also, methods recommended Food sample collection and storage. All foods
for the examination of canned foods by the Food were obtained from local retail outlets. Refrigerated
and Drug Administration (19) involve exposing ground beef was purchased over a 3-month period in
food samples to some oxygen. 454-g plastic tube packages. Refrigerated cheddar
All these methods are generally satisfactory cheese was purchased in 1.6-kg wedges, which were
freshly cut from large cheese wheels and wrapped in
for the recovery of vegetative cells and spores of Saran film.

Downloaded from https://journals.asm.org/journal/aem on 11 March 2022 by 175.176.87.9.

Frozen German hand cheese was pur-
aerotolerant anaerobes and are usually, but not chased in 175-g foil-wrapped packages. All samples
always, adequate for enumeration of clostridia were refrigerated at 7 C and sampled after 1 or 2
in foods. However, methods that allow some days and 1 month.
exposure to oxygen would not be adequate for Food sample preparation. Core samples of the
the recovery of strictly anaerobic bacteria from foods were aseptically transferred and blended in a
foods because these organisms will die if ex- 500-ml Waring blender jar containing enough prere-
posed to oxygen (10). By definition then, anaer- duced anaerobically sterilized (PRAS) medium to
obic methods, such as the Hungate roll-tube yield a 10-fold dilution. The PRAS dilution medium
was of the same composition as that used for culture
technique (10), are necessary to adequately of the specimen, except that it contained no
quantitate the obligately anaerobic organisms The apparatus with core sampler inserted wasagar. con-
in the food, including those organisms that may tinuously flushed with oxygen-free CO2 (Fig. 1).
be injured or debilitated (15). Thus, in this Media composition and preparation. The PRAS
study, roll-tube techniques and habitat-simu- media were prepared using methods described by
lating media were used to isolate and charac- Hungate (10) and were dispensed into 25- by 142-mm
terize anaerobic bacteria present in three foods: roll tubes for enumeration studies and into 16- by
ground beef, cheddar cheese, and German hand 150-mm tubes for stock culture maintenance. Tubes
cheese. were sealed with butyl rubber stoppers. Screw-cap
A preliminary report of this work was pre- septaroll tubes (18 by 142 mm), sealed with butyl rubber
sented earlier (W. M. Gray and M. G. Johnson, tubes (1, 12) were used for fermentation studies. All
were obtained from Bellco Glass, Inc., Vine-
Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, land, N.J.
E104, p. 18). The basal medium (designated PYG) consisted of
' Technical contribution no. 1297 of the South Carolina 1% (wt/vol) each of peptone (Difco Laboratories, De-
Agricultural Experiment Station, Clemson University, troit, Mich.), yeast extract (Difco), and glucose, 2%
Clemson, S.C. 29631. (vol/vol) each of inorganic salt solutions A and B
268
VOL. 31, 1976
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-
OBLIGATE ANAEROBES FROM CHEESE 269
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).
Downloaded from https://journals.asm.org/journal/aem on 11 March 2022 by 175.176.87.9.
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
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
TABLE 1. Ability of isolates obtained from foods by anaerobic or aerobic methods to grow as obligate
anaerobes, facultative anaerobes, or aerobes
Food Isolation Culturable count/g
method (range)
Ground beef Aerobic 6.5 x 104-1.4 x 10W
Anaerobic 1.7 x 10:$-3.8 x 10o
Cheddar cheese Aerobic
Anaerobic 7.9 x 10'i-2.5 x 10f
6.3 x 106-1.3 x 10?
German hand cheese Aerobic
Anaerobic 1.6 x 107-2.0 x 107
1.6 x 10X-2.5 x 107
APPL. ENVIRON. MICROBIOL.
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
Downloaded from https://journals.asm.org/journal/aem on 11 March 2022 by 175.176.87.9.
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-
No. of iso- Isolates (%) that were:
lates exam- Obligate Facultative Obligate
ined
aerobes anaerobes anaerobes
1,030 50 50 0
182 0 100 0
220 5 95 0
213 0 91 9
119 21 79 0
53 0 100 0
VOL. 31, 1976
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-
OBLIGATE ANAEROBES FROM CHEESE 271
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

Downloaded from https://journals.asm.org/journal/aem on 11 March 2022 by 175.176.87.9.

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).
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
APPL. ENVIRON. MICROBIOL.
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
Downloaded from https://journals.asm.org/journal/aem on 11 March 2022 by 175.176.87.9.
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
VOL. 31, 1976
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.
LITERATURE CITED
1. Attebery, H. R., and S. M. Finegold. 1969. Combined
screw-cap and rubber-stopper closure for Hungate
tubes (pre-reduced anaerobically sterilized roll tubes
and liquid media). Appl. Microbiol. 18:558-561.
2. Breed, R. S., E. G. D. Murray, and N. R. Smith (ed.).
1957. Bergey's manual of determinative bacteriology,
7th ed. The Williams & Wilkins Co., Baltimore.
3. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Ber-
gey's manual of determinative bacteriology, 8th ed.
The Williams & Wilkins Co., Baltimore.
4. Claybaugh, G. A. 1954. Non-sporulating obligately an-
aerobic bacteria in dairy products. Iowa State Coll. J.
OBLIGATE ANAEROBES FROM CHEESE 273
Sci. 28:293-295.
5. Dowell, V. R., Jr., and T. M. Hawkins. 1974. Labora-
tory methods in anaerobic bacteriology, CDC labora-
tory manual. U.S. Department of Health, Education,
and Welfare, Public Health Service, Center for Dis-
ease Control, Atlanta.
6. Gibbs, B. M., and B. Freame. Methods for the recovery
of clostridia from foods. J. Appl. Bacteriol. 28:95-111.
7. Hettinga, D. H., G. W. Reinbold, and E. R. Vedamu-
thu. 1974. Split defect of Swiss cheese. I. Effect of
strain of Propionibacterium and wrapping material.
J. Milk Food Technol. 37:322-328.
8. Hirsh, A., and E. Grinsted. 1954. Methods for the
growth and enumeration of anaerobic spore-formers
from cheese, with observations on the effect of nisin.
J. Dairy Res. 21:101-110.
9. Holdeman, L. V., and W. E. C. Moore (ed.). 1972.
Anaerobe laboratory manual. Virginia Polytechnic
Institute and State University Anaerobe Laboratory,
Blacksburg.
10. Hungate, R. E. 1969. A roll tube method for cultivation
of strict anaerobes, p. 117-132. In J. R. Norris and D.
W. Ribbons (ed.), Methods in microbiology, vol. 3B.
Academic Press Inc., New York.
11. Lederberg, J., and E. M. Lederberg. 1952. Replica plat-
ing and indirect selection of bacterial mutants. J.
Bacteriol. 63:399-406.
12. Macy, J. M., J. E. Snellen, and R. E. Hungate. 1972.
Use of syringe methods for anaerobiosis. Am. J. Clin.
Nutr. 25:1318-1323.
13. Moore, W. E. C., and L. V. Holdeman. 1974. Genus I.
Propionibacterium, p. 633-641. In R. E. Buchanan and
N. E. Gibbons (ed.), Bergey's manual of determina-
tive bacteriology, 8th ed. The Williams & Wilkins
Co., Baltimore.
14. Mossel, D. A. A., A. S. DeBruin, H. M. J. Van Diepen,
C. M. A. Vendrig, and G. Zoutewelle. 1956. The enu-
meration of anaerobic bacteria, and of Clostridium
species in particular, in foods. J. Appl. Bacteriol.
19:142-154.
15. Pierson, M. D., S. L. Payne, and G. L. Ades. 1974. Heat
injury and recovery of vegetative cells of Clostridium
Downloaded from https://journals.asm.org/journal/aem on 11 March 2022 by 175.176.87.9.
botulinum type E. Appl. Microbiol. 27:425-426.
16. Prevot, A. R. 1966. Manual for the classification and
determination of the anaerobic bacteria. Lea and Fe-
biger, Philadelphia.
17. Pulverer, G., and H. L. Ko. 1973. Fermentative and
serological studies on Propionibacterium acnes. Appl.
Microbiol. 25:222-229.
18. Rogosa, M. 1974. Genus II. Peptostreptococcus, p. 522-
525. In R. E. Buchanan and N. E. Gibbons (ed.),
Bergey's manual of determinative bacteriology, 8th
ed. The Williams & Wilkins Co., Baltimore.
19. U.S. Department of Health, Education, and Welfare.
1972. Bacteriological analytical manual for foods, 3rd
ed. Public Health Service, Food and Drug Adminis-
tration, U.S. Department of Health, Education, and
Welfare, Washington, D.C.