Comparison identification

V. R. Dowel!, Jr., Ph.D.

of techniques of anaerobic

for isolation bacteria2

and

In this symposium a variety of systems for cultivation of anaerobic bacteria have been described and the advantages of some of the recently described culture techniques have been clearly shown. However, there is litfie published information on comparison of these systems, and it is difficult for microbiologists with limited experience in anaerobic bacteriology to choose which system(s) are most suitable for their laboratories. The choice is particularly difficult for a hospital bacteriologist in a busy clinical laboratory with limited space and funds for anaerobic bacteriology. During the last several years, we have evaluated various systems for cultivation and characterization of anaerobes in our laboratory and the results of some of these studies are summarized in this report. Materials
in use

Comparison of anaerobic systems recovery of anaerobic bacteria (7) In this study, an anaerobic glove by

for

quantitative box (Fig. 3),

similar
the (4)

to the one

described

standard Brewer were compared

jar (2), with respect

Aranki et a!. (8), and the GasPak jar

to rapidity of

growth

and

quantitative

of Clostridium Clostridium haemolyticum, Propionibacterium acnes, Bacteroides fragilis, and Fusobacterium nucleatum (F. fusiforme) obtained from the collection of the Center for Disease Control (CDC) Anaerobe Laboratory. Dilutions of cultures were prepared in

recovery of stock strains perfringens, Clostridium tertium, novyi, types A and B, Clostridium

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NIH thioglycolate selected dilutions

and
evaluation

methods
studies

Some

of the

systems

for

cultivation

of anaer-

obes were evaluated on an in use basis by comparing the growth of stock cultures and reference diagnostic cultures on the surface of blood agar in the test system with that obtained in a Brewer jar (I) (Fig. 1), as described by Dowell and Hawkins (2). Systems evaluated in this manner included

broth within the glove box and were plated in quadruplicate on fresh (prepared not more than 3 hr prior to use) blood agar (trypticase soy agar, BBL, with 0.5% yeast extract, Difco, and 5% defibrinated rabbit blood added) and on the same medium that had been allowed to reduce for 48 hr in the glove box before use. The diluted bacterial cultures were then removed from the glove box, plated in quadruplicate on fresh and prereduced blood agar, and placed immediately into the GasPak and Brewer jars. The plates in the jars were incubated in a conventional incubator at 35 to 37 C and those in the glove box were incubated at the same temperature in an incubator within the glove box. Comparison of anaerobic (4), of anaerobic systems bacteria from clinical for isolation specimens (9) 3), the GasPak

the original tor for use self-contained

GasPak disposable hydrogen generain the standard Brewer jar (3), the carbon dioxide-hydrogen anaerobic

system (GasPak) (Fig. 2) designed by Brewer and Allgeier (4), a disposable anaerobic system (5), and the Torbal jar (6). We also compared the growth of anaerobes in a Case jar using a gas mixture (80% N2, 10% H2, 10% C02) and a room temperature DEOXO catalyst as recommended (per-

jar with prereduced, anaerobically sterilized (PRAS) medium (Robbin Laboratories, Chapel Hill, N.C.) inoculated with a VP! Anaerobic Culture System (Bellco) (Fig.
An anaerobic glove box (Fig. and a roll-streak tube system 4) were compared teria from selected
materials were

for recovery of clinical specimens.

from the

anaerobic bacThe clinical

Memorial

obtained

Grady

Hospital

in Atlanta,

Georgia

and

only

specimens

sonal

communication,

Clarence

Hall,

Washington

Department of Health Laboratories, Seattle) and in a vacuum desiccator (using the same gas mixture and room temperature catalyst) with that obtained in the standard Brewer jar (2). None of these studiees were quantitative and the rapidity of growth was not recorded, as the jars were not opened until after 48 hr of incubation at 35 to
37 C. The

From the Laboratory Division, Center for Disease Control, Health Services and Mental Health Administration, U. S. Department of Health, Education and Welfare, Atlanta, Georgia 30333. Use of trade names is for identification only and does not constitute endorsement by the Health Services and Mental Health Administration, or by the U.S. Department of Health, Education and

Welfare.
American
Journal

of Clinical

Nutrition

25:

DECEMBER

1972,

pp.

1335-1343.

Printed

in U.S.A.

1335

1336

DOWELL

STICK

GAS

2-STAGE REGULATOR

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GAS

TANK

(or

T-TUBE (TEFLON) BRASS)

TUBING

ANAEROBE JAR INDICATOR

VACUUM

SOURCE

FIG. 1. Diagram

of the anaerobic

system

employing

a standard

Brewer

jar

as described

by Dowell

and

Hawkins

(2).

from patients with brain, pelvic, intraabdominal, or other deep abscesses were included in the study. Material from surgically exposed abscesses was aspirated with a syringe and needle, cleared of air, and immediately injected into a sterile gassed out anaerobic collection tube. The specimen tube
was then transported at ambient temperature, to

ber

the CDC Anaerobe Laboratory for processing. The time in transit was normally less than 1 hr. The anaerobic collection tubes were prepared as follows: 1 ml of resazurin agar (2% Difco agar
with 0.0002% resazurin) (15 by 125 mm, screw was added to each tube cap), the tubes were loosely

covered mm at

with

aluminum

foil,

autoclaved

for

15

121 C, and then placed in an anaerobic glove box containing an atmosphere of 85% N2, 5% C02, 10% H,, and less than 10 ppm oxygen, as measured with an oxygen analyzer (Model GP,

Lockwood and McLorie, Inc., P.O. Box 113, Hirsham, Pa. 19044). After the resazurin agar was reduced, the tubes were left in the glove box for an additional 24 to 48 hr, plugged with sterile rub-

stoppers, and screw caps were fastened securely before removal of the tubes from the anaerobic environment. If the stoppers were not secure when the tubes were removed, the resazurin agar turned pink in less than S mm. When the clinical specimen arrived in the CDC Anaerobe Laboratory, the collection tube was placed in the anaerobic glove box immediately and the specimen was mixed to obtain a homogeneous suspension. If the material was viscous, a small quantity (approximately 1 part broth plus 1 part specimen) of NIH thioglycolate broth (Difco) was added before mixing. The resazurin agar under the specimen remained colorless during the mixing procedure. Smears for Gram stain were prepared, and plates of various media were inoculated with a 0.01 ml platinum-rhodium bacteriologic loop (Arthur H. Thomas, Philadelphia, Pa., catalog No. 7012-F20). Five plating media, designated as prereduced (PR) media, were prepared and dispensed in the regular manner, but as soon as the media were

COMPARISON

OF

CULTURE

TECHNIQUES

1337

solidified,

the plates

were

placed

in the glove

box

and held under anaerobic conditions at ambient temperature for a minimum of 48 hr before use as described by Aranki et a!. (8). The media used

were: Trypticase Soy Agar (PR-TSYEA, BBL) plus yeast extract, 5% defibrinated rabbit blood, S g/ml hemin, and 0.5 1ug/ml menadione (2). 2) Neomycin Vancomycin Agar (PR-NVA), the same as TSYEA plus 100 ,ug/ml neomycin, and
1)

0.5%

7.5 g/ml vancomycin. 3) Phenylethyl Alcohol Agar (PR-PEA) (BBL) plus S ig/ml hemin, 0.S jg/ml menadione, and 5% defibrinated rabbit blood. 4) Schaedler Agar (PR-SA) (BBL) plus 0.5 /.Lg/ ml menadione and 5% defibrinated rabbit blood. 5) Brain Heart Infusion Agar (PR-BHIA) plus 0.5 mg/ml cysteine (to equal the concentration of cysteine in the PRAS-BHIA). In addition to the PR media, fresh TSYEA medium (prepared and held in air no longer than 1 hr before use) was used. A plate of each of these media was inoculated with one loopful of
the clinical specimen and streaked to obtain iso-

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FIG.
scribed

2. Photograph
by Brewer and

of
Allgeier

GasPak
(4).

(BBL)

jar

de-

lated colonies. Glass Petri dishes (100 by 15 mm) with metal covers containing absorbent discs (BBL) were used with all plating media. After plating in the glove box was completed,
the specimen tube was re-stoppered, removed from

FIG. 3. Photograph
et at. (8).

of anaerobic

glove

box used

in the study which issimilar

to the one described

by

Aranki

1338

DOWELL

Pak jars, candle jars) were incubated in a conventional bacteriological incubator, all at 35 to
37 C.

tubes and plates in the glove box inspected daily for bacterial colonies. After 48 hr the GasPak jars were placed in the glove Roll-streak

were
box,

opened,

and

the

plates

were

inspected

under

anaerobic

conditions.

The colonial characteristics of all colony types on each medium were described with the aid of a 3.5 X double lens magnifier or a stereoscopic microscope, and a Gram-stained smear of the cells in each was examined microscopically. An isolated colony of each colony type was subcultured to two BHIA (BBL) slants and/or two slants of PR-TSYEA and a tube of peptone-yeast-extractglucose (PYG) broth (11). One slant of each medium was removed and incubated aerobically to determine the oxygen tolerance and purity of the isolate, and the other slants were incubated in the glove box. Pure culture isolates of anaerobes were identified with differential tests described by Dowell and Hawkins (2) and Cato et al. (10). End products in

Downloaded from www.ajcn.org by on June 22, 2009

PYG broth were determined by gas-liquid chromatography as described by Dowel! and Thompson (11). Aerobic and facultative bacteria were identified with media and methods described in
Diagnostic Procedures for Bacterial, Mycotic and Parasitic infections (12). All primary isolation media were discarded after 2 weeks incubation if there was no bacterial growth or if no new colony types appeared.

Results In
FIG. Culture
4. Photograph of the Beilco VP!

use

evaluation

studies

Anaerobic

System.

the glove box, and placed in a Beilco VP! Anaerobic Culture System under a flow of oxygenfree gas (97% CO2, 3% H2) passed through a DEOXO Catalytic Purifier (Englehard Industries Gas Equipment Division, East Newark, NJ.). Two roll-streak tubes of PRAS-BHIA (Robbin Laboratories) were then inoculated as described by Cato et al. (10) with 1 loopful of clinical material per tube. The specimen was then removed from the Be!lco

It was found that all of the anaerobic systems (GasPak jar with carbon dioxide-hydrogen generator, disposable anaerobic system, Torbal jar, Case jar, vacuum desiccator) except the original GasPak hydrogen generator (3) were satisfactory for cultivation of the commonly encountered anaerobes if the jars were used as described and the catalyst to remove residual oxygen was active.

apparatus
TSYEA,

and

a plate

of each

PR

medium

(PR-

PR-NVA, BHIA) and a plate lated with a loopful

for isolation,
as possible.

and

PR-PEA, PR-SA, and PRof fresh TSYEA were inocuof clinical material, streaked placed in GasPak jars as rapidly TSYEA and a plate of Macinoculated in a similar manner candle jar for isolation of aerbacteria. in the anaerobic glove box an incubator within the chammedia (roll-streak tubes, Gas-

A plate of fresh Conkey agar were and incubated in a obic and facultative Media inoculated were incubated in ber and the other

In our experience, use of an inactive catalyst and oxidized media were the two most common causes of failure to cultivate cultures of anaerobes. For these reasons, we employ fresh or prereduced plating media and replace the catalyst in jars with new or rejuvenated catalyst at frequent intervals. We now rejuvenate the catalyst pellets for the GasPak jar by heating at 160 to 170 C in a dry heat oven for 2 hr and the catalyst in the lid of the jar is replaced each time the jar is used (suggestion of Dr. Vera Sutter,

COMPARISON

OF

CULTURE

TECHNIQUES

1339

personal communication). Brewer and Allgeier (4) mentioned that hydrogen sulfide, chlorine, and sulfur dioxide gases will poison the catalyst used in the GasPak jar. The availability of room temperature catalyst in bulk (Engeihard Industries Gas Equipment Division, East Newark, N.J.) allows the use of various types of containers (any that can be evacuated and replaced with a gas mixture containing hydrogen) for cultivation of
anaerobic bacteria.

not shown in Table 1, this was also true C. haemolyticum. It was demonstrated in a separate study by Starr et a!. (13) that blood agar prepared with Schaedlers medium (BBL) is superior to TSYEA for recovery of C. novyi type B and C. haemolyticum. The factors responsible for increased recovery have not been elucidated entirely but the presence of cysteine in reduced Schaedlers medium apparently plays a role (S. E. Starr and V. R. Dowell, Jr., unpublished data). are

for

Comparison recovery

of anaerobic systems of anaerobic bacteria

for Comparison of anaerobic isolation of anaerobic clinical specimens systems bacteria for from

Representative data from the comparison of the recovery of representative anaerobes in the glove box, GasPak, and the Brewer jar systems with fresh and prereduced media are shown in Table 1. With few exceptions, recovery of anaerobes in the GasPak and Brewer jars compared favorably with recovery in the glove box. Little or no difference in recovery was noted with C. perfringens, C. terlium, C. novyi type A, P. acnes, B. fragilis, and F. nucleatum. However, marked differences in recovery were noted with C. novyi type B and C. haemolyticum. Recovery of these was much better on plates processed inside the glove box as compared with those processed outside for the GasPak and Brewer jars. Little or no difference was noted in the number of colonies recovered on fresh and prereduced media with the less strict anaerobes. However, as shown in Table 1, greater recovery of C. novyi type B was achieved on prereduced agar than on fresh agar. Although the data
TABLE I jar, Brewer prereduced jar, and anaerobic media

Clinical materials obtained from 15 patients were examined in the comparative study of the anaerobic glove box, GasPak, and roll-streak tube systems. These included four with pelvic abscess, three with peritonitis, two with intraabdominal abscess (origin unknown) and one each with appendiceal abscess, ovarian dermoid cyst, cholecystitis, pulmonary empyema, neck abscess, and epidural abscess. One specimen yielded a pure culture of a single anaerobe, three contained a mixture of anaerobes, seven contained a mixture of anaerobic and facultative bacteria, and no anaerobes or facultative microorganisms were recovered from four of the specimens. A total of 29 anaerobes, representing 15 species or groups, were recovered
from the 15 specimens.

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Of the 29 anaerobes recovered, 28 were isolated in both the anaerobic glove box and the GasPak system, and 23 were recovered

Comparison of GasPak anaerobes on fresh and

glove

box

for

recovery

of

Average GasPak Log dii F Log dil

number Brewer

of colonies

per plate Glove Log dii box

Organism

R
170

C. tertium C. ,zovyi A

5 5

167 65

176

C. novyi B C. haemolyiicum
F
=

5
2 medium. R
=

0
0

66 0
0

5
5 2

41 0
0

147 72

0
0

5 5 5 3

186

211

50 0

81 43 161

fresh

prereduced medium.

1340

DOWELL

TABLE Recovery

2 of anaerobes from clinical specimens with various media

Number

and
of times

anaerobic
isolated

systems

Organism

isolated

GasPak

and

glove

box

systems

Roil-sreak All systems

PR-SA

TSYEA

TSYEA

BHIA

All media

Bacteroidesfragilis B. melaiiinogenicus
B. incommunis

Bacteroides species Fusobacterium iuc1eaturn F. izecrophorum F.moriiferum

Peptostreptococcus

3 1 1 la 2 1 2 2a 0 2 1 1 1 1 23

4 1 1 2 2 1 2 4 0 1 1 1 1 1 26

4 1 1 2 2 1 2 4 0 1 1 1 1 1 26 TSYEA from
=

0 1 1 2 2 1 1 3 0 1 1 1 1 1 20

4 1 1
3b

0 1 1
2

4 1 1
3b

2 1 2 4 0 2 1 1 1 1 28

2 1 2 4 1 1 1 1 1 1 23
=

2 1 2 4 1 2 1 1 1 1 29

CDCgroupl CDC group CDC group Clost ridium C. sphenoides C.cadaveris Clostridium Propionibacterium Total Legend:
heart infusion
#{176} PR-SA

2 3 perfringeizs

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species acizes

PR was

prereduced;
PRAS
=

agar;

SA Schaedler agar; prereduced, anaerobically

=

trypticase soy agar; BHIA which two

brain

sterilized. isolated once from

group 2 peptostreptococci NVA plates but was not

not

inoculated

and one F. zucleaium were recovered with nonselective

with isolated.

the
b

first two specimens F. nucleatun was

media. numbered the F. nucleatum on nonselective media. One C. perfringens was isolated only on PR-SA on which a small number of hemolytic colonies were detected in an area of confluent growth on the primary plate. Four B. melaninogenicus were detected with TSYEA, PR-TSYEA, PR-SA, but not with PR-BHIA. Twenty-three anaerobes were recovered with the PRAS-BHIA roll-streak tubes and only 20 with PR-BHIA in the anaerobic glove box and GasPak systems. The three organisms not recovered on PR-BHIA were all peptostreptococci, two of which were isolated on other media in both the GasPak and the glove box systems. There was no difference in the results obtained with fresh TSYEA and PR-TSYEA. Discussion These
anaerobic vation studies systems have are shown satisfactory

with the roll-streak system. Four Bacteroides melaninogenicus, one Fusobacterium nudeatum, and one strain of Clostridium perf ringens (in small numbers) were isolated in the glove box and the GasPak jar but not in the roll-streak tubes. On the other hand, one Peptostreptococcus species CDC group 3 was recovered in a roll-streak tube but not in the anaerobic glove box or GasPak jar (Table 2). Table 2 also shows the number of times that various anaerobes were recovered on different media with the anaerobic systems employed. The results for the glove box and GasPak are shown together because they were identical for each system. Results for recovery of anaerobes with PR-NVA and PR-PEA are not shown in Table 2, as only one organism (F. nucleatum) was recovered with these selective media that was not isolated with nonselective media. This specimen contained a mixture of F. nucleatum and a Peptostreptococcus species that greatly out-

that
for

various
culti-

of

anaerobes

commonly

associated

COMPARISON

OF

CULTURE

TECHNIQUES

1341

with human infections, principles of anaerobic lowed. These include:

and handling mize exposure use of fresh

provided that certain bacteriology are fol1) proper collection

a PRAS
mary

they

fluid medium (E medium) for priculture instead of roll-streak tubes and did not use prereduced or fresh blood

of clinical specimens to minito atmospheric oxygen, 2) or prereduced media, and 3)

proper use of the anaerobic system, providing an active catalyst to allow removal of residual oxygen, is particularly important. In general, the glove box roll-streak tube and the GasPak jar, as used in the comparative study, were comparable in recovery of commonly encountered respect to anaer-

agar as we did in our study. Also, 12 of the clinical specimens examined in their study were from sources readily contaminated with anaerobes of the normal flora (e.g., sputum,

urine, In

ear, skin, and vagina). our study of selected

specimens,

pri-

marily from deep abscesses, the GasPak system performed comparably with the rollstreak tube system in recovery of moderately

obes. The absence of hemin and menadione in the BHIA explains the failure to recover B. melaninogenicus from the PRAS-BHIA
roll-streak tubes or the PR-BHIA in the

oxygen-sensitive anaerobes as defined by Loesche (16). However, the GasPak system was used under optimal conditions in our
study by changing the catalyst each time a jar was used, employing fresh or prereduced plating media, and by limited exposure of clinical specimens to atmospheric oxygen during collection and plating procedures. Also, the bacterial colonies obtained in the

glove box and GasPak systems. Cato et al. (10) recommended the use of blood agar (incubated in an anaerobic jar) and other media
in conjunction PRAS-BHIA and cocci with the roll-streak tubes of for isolation of anaerobic rods from clinical specimens. The

Downloaded from www.ajcn.org by on June 22, 2009

GasPak jars were inspected in an anaerobic glove box,

further limited exposure

and subcultured a procedure that

anaerobes to

BHIA-Brain
mented presently

Heart

Infusion
recommended

Agar-Suppleby the VPI

of the

atmospheric

oxygen.

This

was

done

Anaerobe Laboratory (14) contains brain heart infusion agar supplemented with yeast extract, heme, and vitamin K (menadione). The handling of clinical specimens before inoculation of media appeared to be a critical factor
organisms specimens,

in recovering
were recovered

anaerobes.
from

three

bacteria

were

although observed

organisms in direct

microof the resembling smears of

the resaz-

No

the original samples. In one case urin indicator in the collection distinctly pink when the specimen

sure subculture and identification aerobes that had grown on the primary isolation media in the GasPak jar. It would be difficult to use the GasPak jar in such a way in a busy clinical microbiology laboratory. The results obtained by Rosenblatt et al. (17) in their excellent, comprehensive study of various anaerobic systems were quite similar to ours. In the study of 24 selected clinical specimens from a variety of different
sites they found

to inof all an-

tube was was re-

that

the GasPak

jar was

as

effective

ceived at CDC, suggesting that the specimen was oxidized. Transportation of one specimen was delayed several hours and there was no obvious explanation for failure to recover organisms from the third specimen. Our results did not agree McMinn and Crawford (15) that commercially prepared with those of who reported PRAS media

as the other more complex methods (glove box, roll-streak tube, et cetera) for qualitative and quantitative recovery of anaerobes. Organisms isolated included B. fragilis, B. melaninogenicus, F. nucleatum (F.

used
workers

as

recommended

by

Moore

and

co-

of the VPI Anaerobe Laboratory were superior to thioglycolate broth or conventional blood agar plates incubated in the

fusiforme), B. oralis, P. acnes, Sphaerophorus necrophorus, Eubacterium species, C. perfringens and Peptostreptococcus asaccharolyticus. They also emphasized the importance of proper collection and transportation of clinical specimens in the recovery of anaerobic bacteria.

GasPak
from

system
clinical most

recovery of specimens. It should

instances these

for

anaerobes be noted
used

Each of the anaerobic systems (GasPak jar, roll-streak tube system, anaerobic glove box) has advantages and disadvantages. The
GasPak system is readily available commer-

that in

workers

1342

DOWELL

cially, single

relatively little space (for a is quite simple to use. However, it must be opened in order to allow examination of cultures and this is a major disadvantage because the colonies are exposed to atmospheric oxygen. Early or frequent observation of plates is impractical because the jars should not be opened for at least 48 hr after inoculation of media with clinical materials. This important consideration is frequently neglected in clinical laboratories apparently, as evidenced by the small number of GasPak jars in use as compared with the specimen workload. When a sufficient number of GasPak jars to accommodate the workload is used, the cost involved is similar to that for an anaerobic glove box and greater than that for the Beilco VPI Anaerobe Culture System. It is well known that some strict anaerobes, as defined by Loesche (16), will not tolerate exposure to oxygen. None of the strict anaerobes listed by Loesche (Treponema macrodentium, Treponema denticola, Treponema oralis n. sp., Clostridium haemolyticum, Selenomonas ruminantiun2, Butyrivibrio fibrisolvens, Succinivibrio dextrinosolvens, and Lachnospira multiparus) were recovered by us from clinical materials or by Rosenblatt et al. (17) in their study of selected clinical specimens. The anaerobic glove box is now available

requires jar) and

ceptibility of anaerobes by the disc method. On the other hand, the roll-streak system is excellent for quantitative studies of anaerobic bacteria, e.g., in intestinal contents, saliva, et cetera.

Conclusion
The systems oratory technical choice of which anaerobic system or to use in a clinical microbiology labshould be made on the basis of the capabilities of the laboratory personnel, the availability of space for anaerobic bacteriology, the specimen workload, the need for special studies requiring anaerobic techniques, and various other factors. If the laboratory personnel are deficient in their knowledge of anaerobic bacteriology, the first investment, before purchasing equipment, should be to correct the gap in knowledge. With an adequate knowledge of the
principles vantages of of anaerobic sophisticated bacteriology, equipment the such adas

Downloaded from www.ajcn.org by on June 22, 2009

the VPI anaerobic

Anaerobic glove box

Culture System and will become evident.

the

References
1.
BREWER,

J. H.

anaerobe
2.

jar.
V.

I.
R.,

A modification Lab. Clin.

JR.,

of the Brown Med. 24: 1190,

1939. DOWELL,
Laboratory ogy. Public

commercially, but not on a large-scale basis. It is somewhat more difficult to use than the GasPak jar and requires more space, which
can be a major disadvantage in some laboratories. The necessity for working with gloves is also a disadvantage but is easily overcome with practice. The major advantage of the glove box system and the roll-streak tube system is that cultures can be examined and manipulated at will without exposing them to high levels of atmospheric oxygen. The Belico VPI Anaerobic Culture System as well as PRAS media of various types are available commercially. The VPI Anaerobic Culture System requires little space for operation but it is considered by some to be more difficult to use than either the GasPak or the glove box systems. In addition, it is difficult to use an opaque medium with the roll-streak tube system and the system is not
suitable for determining

Methods

Health Washington, D. C.: Office, 1968. 3. BREWER, J. H., Arm D. able hydrogen generator.
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AND T. M. HAWKINS. in Anaerobic BacteriolService Publ. No. 1803, U.S. Government Printing

L.

ALLGEIER.

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WILLIS,

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G. E., S. E. STARR AND D. N. Comparison of anaerobic systems for recovery of anaerobic bacteria. Bacieriol. Proc. 1971, p. 108. 8. AaANKI, A., S. A. SYED, E. B. KENNEY AND R. FRETER. !solation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. 4ppl. Microbiol. 17: 586, 1969.
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DEL-

TECHNIQUES

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KILLOORE AND V. R. of Schaedler agar extract agar for the bacteria. App!. Mi-

9.

KILLGORE,

G.
N.

E.,
WHALEY

S.

E.
AND

STARR,

V.

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STARR, DOWELL,

S.

V. R. DowELL, Comparison of three anaerobic systems the isolation of anaerobic bacteria from ical specimens. Abstracts Ann. Meeting Soc. Microbiol. 1972, p. 94.
BENE, D. 10.

JR.

for cliiiAm.
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crobiol.

JR. trypticase

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CATO, E. P., C. S. CuimINs, L. V. HOLDEMAN, J. L. JoHNsoN, W. E. C. Mooiui, R. M. SMIBERT

Methods
burg,

L. DS. Srim. in Anaerobic

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AND

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Univ.

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F. S.

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THOMPSON.

L. V., AND W. E. C. MooRE (editors). Anaerobe Laboratory Manual. Blacksburg, Virginia: Virginia Polytech. Inst. and State Univ., 1972, p. 124. 15. MCMINN, M. T., AND J. J. Citwpoiw. Recovery of anaerobic microorganisms from clinical specimens in prereduced media versus recovery by routine clinical laboratory methods.
HOLDEMAN,

Identification of acid and alcohol products of anaerobic bacteria by gas-liquid chromatography. Center for Disease Control Pub!., At12. lanta, Ga., 1971. BODILY, H. L., E. L. UPDYKE 4s.irn J. 0. MASON (editors). Diagnostic Procedures for Bacterial, Mycotic and Parasitic Infections. New York: Am. Public Health Assoc., 1970.

App!. 16.
LOESCHE,

Microbiol.

19: 207,

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1969. 17.
ROSENBLArr, FINEGOLD.

W. J. Oxygen sensitivity bacteria. App!. Microbiol.

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J. E., A. M. FALLON AND S. M. Recovery of anaerobes from clinispecimens. Abstracts Ann. Meeting Am.
Microbiol. 1972, p. 94.

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