comparison oftechniques forisolation and identification

The American Journal of Clinical Nutrition 25: DECEMBER 1972, pp. 1335-1343. Printed in U.S.A. 1335

Comparison of techniques for isolation andidentification of anaerobic bacteria”2

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

In this symposium a variety of systems for

cultivation of anaerobic bacteria have been

described and the advantages of some of therecently described culture techniques havebeen clearly shown. However, there is litfie

published information on comparison of

these systems, and it is difficult for microbi-ologists with limited experience in anaerobic

bacteriology to choose which system(s) aremost suitable for their laboratories. Thechoice is particularly difficult for a hospitalbacteriologist in a busy clinical laboratorywith limited space and funds for anaerobic

bacteriology.During the last several years, we have eval-

uated various systems for cultivation and

characterization of anaerobes in our labora-tory and the results of some of these studiesare summarized in this report.

Materials and methods

“in use” evaluation studies

Some of the systems for cultivation of anaer-obes were evaluated on an “in use” basis by com-paring the growth of stock cultures and referencediagnostic cultures on the surface of blood agar inthe test system with that obtained in a Brewer jar(I) (Fig. 1), as described by Dowell and Hawkins(2). Systems evaluated in this manner includedthe original GasPak disposable hydrogen genera-tor for use in the standard Brewer jar (3), theself-contained carbon dioxide-hydrogen anaerobicsystem (GasPak) (Fig. 2) designed by Brewer andAllgeier (4), a disposable anaerobic system (5), andthe Torbal jar (6). We also compared the growthof anaerobes in a Case jar using a gas mixture(80% N2, 10% H2, 10% C02) and a room tem-perature DEOXO catalyst as recommended (per-sonal communication, Clarence Hall, WashingtonDepartment of Health Laboratories, Seattle) andin a vacuum desiccator (using the same gas mix-ture and room temperature catalyst) with that ob-tained in the standard Brewer jar (2). None ofthese studiees were quantitative and the rapidityof growth was not recorded, as the jars were notopened until after 48 hr of incubation at 35 to37 C.

Comparison of anaerobic systems for quantitativerecovery of anaerobic bacteria (7)

In this study, an anaerobic glove box (Fig. 3),similar to the one described by Aranki et a!. (8),the standard Brewer jar (2), and the GasPak jar(4) were compared with respect to rapidity ofgrowth and quantitative recovery of stock strains

of Clostridium perfringens, Clostridium tertium,Clostridium novyi, types A and B, Clostridiumhaemolyticum, Propionibacterium acnes, Bacte-roides fragilis, and Fusobacterium nucleatum (F.fusiforme) obtained from the collection of theCenter for Disease Control (CDC) Anaerobe Lab-oratory. Dilutions of cultures were prepared in

NIH thioglycolate broth within the glove box andselected dilutions were plated in quadruplicate onfresh (prepared not more than 3 hr prior to use)blood agar (trypticase soy agar, BBL, with 0.5%yeast extract, Difco, and 5% defibrinated rabbitblood added) and on the same medium that hadbeen allowed to reduce for 48 hr in the glove boxbefore use.

The diluted bacterial cultures were then re-moved from the glove box, plated in quadruplicateon fresh and prereduced blood agar, and placedimmediately into the GasPak and Brewer jars.The plates in the jars were incubated in a con-ventional incubator at 35 to 37 C and those inthe glove box were incubated at the same temper-ature in an incubator within the glove box.

Comparison of anaerobic systems for isolationof anaerobic bacteria from clinical specimens (9)

An anaerobic glove box (Fig. 3), the GasPak jar(4), and a roll-streak tube system with prereduced,anaerobically sterilized (PRAS) medium (RobbinLaboratories, Chapel Hill, N.C.) inoculated witha VP! Anaerobic Culture System (Bellco) (Fig.4) were compared for recovery of anaerobic bac-teria from selected clinical specimens. The clinicalmaterials were obtained from the Grady Memorial

Hospital in Atlanta, Georgia and only specimens

‘From the Laboratory Division, Center for Dis-ease Control, Health Services and Mental HealthAdministration, U. S. Department of Health, Edu-cation and Welfare, Atlanta, Georgia 30333.

‘Use of trade names is for identification onlyand does not constitute endorsement by the HealthServices and Mental Health Administration, or bythe U.S. Department of Health, Education andWelfare.

Dow

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ic.oup.com/ajcn/article/25/12/1335/4733260 by guest on 31 O

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2-STAGEGAS REGULATOR

STICK

GAS TANK

T-TUBE(TEFLON)

(or BRASS) TUBING

‘VACUUM SOURCE

1336 DOWELL

ANAEROBEJAR

INDICATOR

FIG. 1. Diagram of the anaerobic system employing a standard Brewer jar as described by Dowell andHawkins (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 tubewas then transported at ambient temperature, tothe CDC Anaerobe Laboratory for processing.The time in transit was normally less than 1 hr.

The anaerobic collection tubes were preparedas follows: 1 ml of resazurin agar (2% Difco agarwith 0.0002% resazurin) was added to each tube(15 by 125 mm, screw cap), the tubes were looselycovered with aluminum foil, autoclaved for 15mm at 121 C, and then placed in an anaerobicglove 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 wasreduced, the tubes were left in the glove box foran additional 24 to 48 hr, plugged with sterile rub-

ber stoppers, and screw caps were fastened se-curely before removal of the tubes from the anaer-obic environment. If the stoppers were not securewhen the tubes were removed, the resazurin agarturned pink in less than S mm.

When the clinical specimen arrived in the CDCAnaerobe Laboratory, the collection tube wasplaced in the anaerobic glove box immediatelyand the specimen was mixed to obtain a homoge-neous suspension. If the material was viscous, asmall quantity (approximately 1 part broth plus 1part specimen) of NIH thioglycolate broth (Difco)was added before mixing. The resazurin agar un-der the specimen remained colorless during themixing procedure. Smears for Gram stain wereprepared, and plates of various media were inoc-ulated with a 0.01 ml platinum-rhodium bacteri-ologic loop (Arthur H. Thomas, Philadelphia, Pa.,catalog No. 7012-F20).

Five plating media, designated as prereduced(PR) media, were prepared and dispensed in theregular manner, but as soon as the media were

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COMPARISON OF CULTURE TECHNIQUES 1337

FIG. 3. Photograph of anaerobic glove box used in the study which issimilar to the one described by Aranki

et at. (8).

FIG. 2. Photograph of GasPak (BBL) jar de-

scribed by Brewer and Allgeier (4).

solidified, the plates were placed in the glove boxand held under anaerobic conditions at ambienttemperature for a minimum of 48 hr before useas described by Aranki et a!. (8). The media usedwere:

1) Trypticase Soy Agar (PR-TSYEA, BBL) plus0.5% yeast extract, 5% defibrinated rabbit blood,S �g/ml hemin, and 0.5 1ug/ml menadione (2).

2) Neomycin Vancomycin Agar (PR-NVA), thesame as TSYEA plus 100 ,ug/ml neomycin, and7.5 �g/ml vancomycin.

3) Phenylethyl Alcohol Agar (PR-PEA) (BBL)plus S �ig/ml hemin, 0.S j�g/ml menadione, and5% 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) plus0.5 mg/ml cysteine (to equal the concentration ofcysteine in the PRAS-BHIA).

In addition to the PR media, fresh TSYEAmedium (prepared and held in air no longer than1 hr before use) was used. A plate of each ofthese media was inoculated with one loopful ofthe clinical specimen and streaked to obtain iso-

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

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Results

1338 DOWELL

Pak jars, candle jars) were incubated in a con-ventional bacteriological incubator, all at 35 to

37 C.

Roll-streak tubes and plates in the glove boxwere inspected daily for bacterial colonies. After48 hr the GasPak jars were placed in the glovebox, opened, and the plates were inspected underanaerobic conditions.

The colonial characteristics of all colony typeson each medium were described with the aid of a3.5 X double lens magnifier or a stereoscopic mi-croscope, and a Gram-stained smear of the cellsin each was examined microscopically. An iso-lated colony of each colony type was subculturedto two BHIA (BBL) slants and/or two slants ofPR-TSYEA and a tube of peptone-yeast-extract-glucose (PYG) broth (11). One slant of each me-dium was removed and incubated aerobically todetermine the oxygen tolerance and purity of theisolate, and the other slants were incubated in theglove box.

Pure culture isolates of anaerobes were identi-fied with differential tests described by Dowell andHawkins (2) and Cato et al. (10). End products inPYG broth were determined by gas-liquid chro-matography as described by Dowel! and Thomp-son (11). Aerobic and facultative bacteria wereidentified with media and methods described inDiagnostic Procedures for Bacterial, Mycotic andParasitic infections (12). All primary isolation me-dia were discarded after 2 weeks incubation ifthere was no bacterial growth or if no new colonytypes appeared.

FIG. 4. Photograph of the Beilco VP! AnaerobicCulture System.

the glove box, and placed in a Beilco VP! Anaer-obic Culture System under a flow of oxygen-free gas (97% CO2, 3% H2) passed through aDEOXO Catalytic Purifier (Englehard IndustriesGas Equipment Division, East Newark, NJ.). Tworoll-streak tubes of PRAS-BHIA (Robbin Labora-tories) were then inoculated as described by Catoet al. (10) with 1 loopful of clinical material pertube.

The specimen was then removed from the Be!lcoapparatus and a plate of each PR medium (PR-TSYEA, PR-NVA, PR-PEA, PR-SA, and PR-BHIA) and a plate of fresh TSYEA were inocu-lated with a loopful of clinical material, streakedfor isolation, and placed in GasPak jars as rapidlyas possible.

A plate of fresh TSYEA and a plate of Mac-Conkey agar were inoculated in a similar mannerand incubated in a candle jar for isolation of aer-obic and facultative bacteria.

Media inoculated in the anaerobic glove boxwere incubated in an incubator within the cham-ber and the other media (roll-streak tubes, Gas-

“In use” evaluation studies

It was found that all of the anaerobic sys-tems (GasPak jar with carbon dioxide-hy-drogen generator, disposable anaerobicsystem, Torbal jar, Case jar, vacuum desic-

cator) except the original GasPak hydrogengenerator (3) were satisfactory for cultivationof the commonly encountered anaerobes if

the jars were used as described and the cat-alyst to remove residual oxygen was active.

In our experience, use of an inactive catalyst

and oxidized media were the two most com-mon causes of failure to cultivate cultures ofanaerobes. For these reasons, we employ

fresh or prereduced plating media and re-place the catalyst in jars with new or re-juvenated catalyst at frequent intervals.

We now rejuvenate the catalyst pelletsfor the GasPak jar by heating at 160 to 170C in a dry heat oven for 2 hr and the catalystin the lid of the jar is replaced each time thejar is used (suggestion of Dr. Vera Sutter,

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F = fresh medium. R = prereduced medium.

personal communication). Brewer and All-geier (4) mentioned that hydrogen sulfide,chlorine, and sulfur dioxide gases will poisonthe catalyst used in the GasPak jar. The

availability of room temperature catalyst in

bulk (Engeihard Industries Gas EquipmentDivision, East Newark, N.J.) allows the useof various types of containers (any that can

be evacuated and replaced with a gas mix-ture containing hydrogen) for cultivation ofanaerobic bacteria.

Comparison of anaerobic systems forrecovery of anaerobic bacteria

Representative data from the comparisonof the recovery of representative anaerobesin the glove box, GasPak, and the Brewerjar 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 re-

covery in the glove box. Little or no differ-ence in recovery was noted with C. per-fringens, C. terlium, C. novyi type A, P.

acnes, B. fragilis, and F. nucleatum. How-ever, marked differences in recovery werenoted with C. novyi type B and C. haemo-

lyticum. Recovery of these was much better

on plates processed inside the glove box ascompared with those processed outside forthe GasPak and Brewer jars. Little or no

difference was noted in the number of col-onies recovered on fresh and prereduced me-dia with the less strict anaerobes. However,as shown in Table 1, greater recovery of C.novyi type B was achieved on prereducedagar than on fresh agar. Although the data

TABLE I

are not shown in Table 1, this was also truefor C. haemolyticum.

It was demonstrated in a separate study

by Starr et a!. (13) that blood agar preparedwith Schaedler’s medium (BBL) is superior

to TSYEA for recovery of C. novyi type B

and C. haemolyticum. The factors responsi-ble for increased recovery have not been elu-cidated entirely but the presence of cysteinein reduced Schaedler’s medium apparentlyplays a role (S. E. Starr and V. R. Dowell,Jr., unpublished data).

Comparison of anaerobic systems for

isolation of anaerobic bacteria from

clinical specimens

Clinical materials obtained from 15 pa-tients 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 periton-

itis, two with intraabdominal abscess (ori-gin unknown) and one each with appendicealabscess, ovarian dermoid cyst, cholecystitis,

pulmonary empyema, neck abscess, and epi-dural abscess. One specimen yielded a pure

culture of a single anaerobe, three containeda mixture of anaerobes, seven contained a

mixture of anaerobic and facultative bacte-ria, and no anaerobes or facultative micro-organisms were recovered from four of the

specimens. A total of 29 anaerobes, repre-senting 15 species or groups, were recoveredfrom the 15 specimens.

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

Comparison of GasPak jar, Brewer jar, and anaerobic glove box for recovery ofanaerobes on fresh and prereduced media

Organism

Average number of colonies per plate

GasPak Brewer Glove box

Logdii

F R Logdil F R

Logdii F R

C. tertium

C. ,zovyi AC. novyi BC. haemolyiicum

5

5

52

167

65

00

170

6600

5

55

2

176

4100

147

72

00

5

5

53

186

500

211

8143

161

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1340 DOWELL

TABLE 2

Recovery of anaerobes from clinical specimens with various media and anaerobic systems

Organism isolated

Number of times isolated

GasPak and glove box systems Roil-s�reak

All systems

PR-SA TSYEATSYEA BHIA All media

BacteroidesfragilisB. melaiiinogenicusB. incommunis

Bacteroides speciesFusobacterium �iuc1eaturnF. izecrophorumF.moriiferumPeptostreptococcus

CDCgrouplCDC group 2CDC group 3Clost ridium perfringeizsC. sphenoidesC.cadaverisClostridium speciesPropionibacterium acizes

Total

4

3

11la21

22a021111

23

4

4

11221

24

01

1111

26

4

4

11221

24

011111

26

4

0

11221

13

011111

20

4

4113b

21

24

021111

28

4

0112

21

24

111111

23

4

4113b

21

24

121111

29

Legend: PR = prereduced; SA = Schaedler agar; TSYEA = trypticase soy agar; BHIA = brainheart infusion agar; PRAS = prereduced, anaerobically sterilized.

#{176}PR-SA was not inoculated with the first two specimens from which two group 2 peptostreptococciand one F. �zucleaium were isolated. b F. nucleatu’n was isolated once from NVA plates but was notrecovered with nonselective media.

with the roll-streak system. Four Bacteroides

melaninogenicus, one Fusobacterium nude-

atum, and one strain of Clostridium perf ring-

ens (in small numbers) were isolated in theglove box and the GasPak jar but not in theroll-streak tubes. On the other hand, onePeptostreptococcus species CDC group 3was recovered in a roll-streak tube but notin 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 systemsemployed. The results for the glove box and

GasPak are shown together because theywere identical for each system. Results forrecovery of anaerobes with PR-NVA and

PR-PEA are not shown in Table 2, as onlyone organism (F. nucleatum) was recoveredwith these selective media that was not iso-

lated with nonselective media. This specimencontained a mixture of F. nucleatum and aPeptostreptococcus species that greatly out-

numbered the F. nucleatum on nonselective

media. One C. perfringens was isolated onlyon PR-SA on which a small number of hemo-lytic colonies were detected in an area of

confluent growth on the primary plate. FourB. melaninogenicus were detected withTSYEA, PR-TSYEA, PR-SA, but not withPR-BHIA. Twenty-three anaerobes were re-covered with the PRAS-BHIA roll-streak

tubes and only 20 with PR-BHIA in the an-aerobic glove box and GasPak systems. Thethree organisms not recovered on PR-BHIAwere all peptostreptococci, two of which were

isolated on other media in both the GasPakand the glove box systems. There was no dif-ference in the results obtained with freshTSYEA and PR-TSYEA.

Discussion

These studies have shown that variousanaerobic systems are satisfactory for culti-

vation of anaerobes commonly associated

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with human infections, provided that certainprinciples of anaerobic bacteriology are fol-lowed. These include: 1) proper collectionand handling of clinical specimens to mini-mize exposure to atmospheric oxygen, 2)

use of fresh or prereduced media, and 3)

proper use of the anaerobic system, provid-ing an active catalyst to allow removal ofresidual oxygen, is particularly important.

In general, the glove box roll-streak tubeand the GasPak jar, as used in the compara-tive study, were comparable in respect torecovery of commonly encountered anaer-

obes. The absence of hemin and menadionein the BHIA explains the failure to recoverB. melaninogenicus from the PRAS-BHIAroll-streak tubes or the PR-BHIA in theglove box and GasPak systems. Cato et al.(10) recommended the use of blood agar (in-cubated in an anaerobic jar) and other mediain conjunction with the roll-streak tubes ofPRAS-BHIA for isolation of anaerobic rodsand cocci from clinical specimens. The

BHIA-Brain Heart Infusion Agar-Supple-mented presently recommended by the VPI

Anaerobe Laboratory (14) contains brainheart infusion agar supplemented with yeastextract, heme, and vitamin K (menadione).

The handling of clinical specimens beforeinoculation of media appeared to be a criti-cal factor in recovering anaerobes. No micro-organisms were recovered from three of thespecimens, although organisms resemblingbacteria were observed in direct smears ofthe original samples. In one case the resaz-

urin indicator in the collection tube wasdistinctly pink when the specimen was re-ceived at CDC, suggesting that the specimen

was oxidized. Transportation of one speci-

men was delayed several hours and there

was no obvious explanation for failure to

recover organisms from the third specimen.

Our results did not agree with those ofMcMinn and Crawford (15) who reportedthat commercially prepared PRAS media

used as recommended by Moore and co-workers of the VPI Anaerobe Laboratorywere superior to thioglycolate broth or con-

ventional blood agar plates incubated in the

GasPak system for recovery of anaerobesfrom clinical specimens. It should be notedthat in most instances these workers used

a PRAS fluid medium (“E” medium) for pri-mary culture instead of roll-streak tubes andthey did not use prereduced or fresh bloodagar as we did in our study. Also, 12 of the

clinical specimens examined in their studywere from sources readily contaminated with

anaerobes of the normal flora (e.g., sputum,urine, ear, skin, and vagina).

In our study of selected specimens, pri-marily from deep abscesses, the GasPak sys-tem performed comparably with the roll-streak tube system in recovery of moderatelyoxygen-sensitive anaerobes as defined byLoesche (16). However, the GasPak systemwas used under optimal conditions in ourstudy by changing the catalyst each time ajar was used, employing fresh or prereducedplating media, and by limited exposure ofclinical specimens to atmospheric oxygenduring collection and plating procedures.Also, the bacterial colonies obtained in the

GasPak jars were inspected and subculturedin an anaerobic glove box, a procedure thatfurther limited exposure of the anaerobes toatmospheric oxygen. This was done to in-

sure subculture and identification of all an-aerobes that had grown on the primary iso-

lation media in the GasPak jar. It would bedifficult 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 studyof various anaerobic systems were quite sim-ilar to ours. In the study of 24 selected clin-ical specimens from a variety of different

sites they found that the GasPak jar was aseffective as the other more complex methods(glove box, roll-streak tube, et cetera) for

qualitative and quantitative recovery of an-aerobes. Organisms isolated included B. fra-

gilis, B. melaninogenicus, F. nucleatum (F.

fusiforme), B. oralis, P. acnes, Sphaero-

phorus necrophorus, Eubacterium species,

C. perfringens and Peptostreptococcus asac-

charolyticus. They also emphasized the im-portance of proper collection and transpor-

tation of clinical specimens in the recovery

of anaerobic bacteria.

Each of the anaerobic systems (GasPakjar, roll-streak tube system, anaerobic glovebox) has advantages and disadvantages. TheGasPak system is readily available commer-

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1342 DOWELL

cially, requires relatively little space (for asingle jar) and is quite simple to use. How-ever, it must be opened in order to allowexamination of cultures and this is a majordisadvantage because the colonies are ex-posed to atmospheric oxygen. Early or fre-quent observation of plates is impractical be-cause the jars should not be opened for at

least 48 hr after inoculation of media withclinical materials. This important considera-tion is frequently neglected in clinical labora-tories apparently, as evidenced by the smallnumber of GasPak jars in use as comparedwith the specimen workload. When a suffi-

cient number of GasPak jars to accommo-date the workload is used, the cost involvedis similar to that for an anaerobic glove boxand greater than that for the Beilco VPI An-

aerobe 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 byLoesche (Treponema macrodentium, Trepo-

nema denticola, Treponema oralis n. sp.,Clostridium haemolyticum, Selenomonas ru-minantiun2, Butyrivibrio fibrisolvens, Succini-vibrio dextrinosolvens, and Lachnospira mul-

tiparus) were recovered by us from clinicalmaterials or by Rosenblatt et al. (17) in theirstudy of selected clinical specimens.

The anaerobic glove box is now availablecommercially, but not on a large-scale basis.It is somewhat more difficult to use than the

GasPak jar and requires more space, whichcan be a major disadvantage in some labora-tories. The necessity for working with gloves

is also a disadvantage but is easily overcomewith practice. The major advantage of theglove box system and the roll-streak tubesystem is that cultures can be examined andmanipulated at will without exposing themto high levels of atmospheric oxygen. The

Belico VPI Anaerobic Culture System aswell as PRAS media of various types are

available commercially. The VPI AnaerobicCulture System requires little space for op-eration but it is considered by some to bemore difficult to use than either the GasPak

or the glove box systems. In addition, it isdifficult to use an opaque medium with the

roll-streak tube system and the system is notsuitable for determining the antibiotic sus-

ceptibility of anaerobes by the disc method.

On the other hand, the roll-streak system isexcellent for quantitative studies of anaero-

bic bacteria, e.g., in intestinal contents, sa-liva, et cetera.

Conclusion

The choice of which anaerobic system orsystems to use in a clinical microbiology lab-oratory should be made on the basis of the

technical capabilities of the laboratory per-

sonnel, the availability of space for anaero-bic bacteriology, the specimen workload, theneed for special studies requiring anaerobic

techniques, and various other factors. If the

laboratory personnel are deficient in theirknowledge of anaerobic bacteriology, thefirst investment, before purchasing equip-

ment, should be to correct the gap in knowl-

edge. With an adequate knowledge of theprinciples of anaerobic bacteriology, the ad-

vantages of sophisticated equipment such as

the VPI Anaerobic Culture System and the

anaerobic glove box will become evident. [�

References

1. BREWER, J. H. A modification of the Brownanaerobe jar. I. Lab. Clin. Med. 24: 1190,

1939.2. DOWELL, V. R., JR., AND T. M. HAWKINS.

Laboratory Methods in Anaerobic Bacteriol-ogy. Public Health Service Publ. No. 1803,Washington, D. C.: U.S. Government PrintingOffice, 1968.

3. BREWER, J. H., Ar�m D. L. ALLGEIER. Dispos-able hydrogen generator. Science 147: 1033,1965.

4. BREWER, J. H., AND D. L. ALLGEIER. Safe self-contained carbon dioxide-hydrogen anaerobicsystem. Appl. Microbiol. 14: 985, 1966.

5. BREWER, J. H., AND D. L. ALLGEIER. A dis-posable anaerobic system designed for fieldand laboratory use. Appl. Microbiol. 16: 848,1968.

6. WILLIS, A. T. Anaerobic Bacteriology in Clin-ical Medicine. London: Butterworths, 1964, p.14.

7. KILLGORE, G. E., S. E. STARR AND D. N.WHALEY. Comparison of anaerobic systems forrecovery of anaerobic bacteria. Bacieriol.Proc. 1971, p. 108.

8. AaANKI, A., S. A. SYED, E. B. KENNEY AND

R. FRETER. !solation of anaerobic bacteriafrom human gingiva and mouse cecum bymeans of a simplified glove box procedure.4ppl. Microbiol. 17: 586, 1969.

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9. KILLGORE, G. E., S. E. STARR, V. E. DEL-

BENE, D. N. WHALEY AND V. R. DowELL, JR.Comparison of three anaerobic systems forthe isolation of anaerobic bacteria from cliii-ical specimens. Abstracts Ann. Meeting Am.Soc. Microbiol. 1972, p. 94.

10. CATO, E. P., C. S. Cui�m�INs, L. V. HOLDEMAN,

J. L. JoHNsoN, W. E. C. Mooiui, R. M. SMI-

BERT �n L. DS. Sr�im. Outline of ClinicalMethods in Anaerobic Bacteriology. Blacks-burg, Virginia: Virginia Polytech. Inst. andState Univ. Anaerobe Laboratory, 1970.

11. DowEu, V. R., JR., AND F. S. THOMPSON.

Identification of acid and alcohol products ofanaerobic bacteria by gas-liquid chromatog-raphy. Center for Disease Control Pub!., At-lanta, Ga., 1971.

12. BODILY, H. L., E. L. UPDYKE 4s.i�rn J. 0. MASON(editors). Diagnostic Procedures for Bacterial,Mycotic and Parasitic Infections. New York:Am. Public Health Assoc., 1970.

13. STARR, S. E., G. E. KILLOORE AND V. R.DOWELL, JR. Comparison of Schaedler agarand trypticase soy-yeast extract agar for thecultivation of anaerobic bacteria. App!. Mi-crobiol. 22: 655, 1971.

14. HOLDEMAN, L. V., AND W. E. C. MooRE (edi-tors). Anaerobe Laboratory Manual. Blacks-burg, Virginia: Virginia Polytech. Inst. andState Univ., 1972, p. 124.

15. MCMINN, M. T., AND J. J. Ci�twpoiw. Re-covery of anaerobic microorganisms from clin-ical specimens in prereduced media versus re-covery by routine clinical laboratory methods.App!. Microbiol. 19: 207, 1970.

16. LOESCHE, W. J. Oxygen sensitivity of variousanaerobic bacteria. App!. Microbiol. 18: 723,1969.

17. ROSENBLArr, J. E., A. M. FALLON AND S. M.FINEGOLD. Recovery of anaerobes from clini-cal specimens. Abstracts Ann. Meeting Am.Soc. Microbiol. 1972, p. 94.

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comparison oftechniques forisolation and identification

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