APPLIED MICROBIOLOGY, Jan. 1973, p. 77-85Copyright 0 1973 American Society for Microbiology

Vol. 25, No. 1Printed in U.SA.

Comparison of Methods for Isolation ofAnaerobic Bacteria from Clinical Specimens

JON E. ROSENBLATT, ANN FALLON, AND SYDNEY M. FINEGOLD

Infectious Disease Section, Medical Service, Veterans Administration Wadsworth Hospital Center, andUniversity of California at Los Angeles School of Medicine 90073

Received for publication 17 May 1972

Five different anaerobic culture methods and several different media werecompared for their ability to recover anaerobes from clinical specimens.Specimens were obtained from patients with documented infections, avoidingcontamination with normal flora, and immediately placed in an anaerobictransporter. Each specimen was cultured by all methods and on all the variousmedia. The comparative data indicate that anaerobic jars (GasPak andevacuation-replacement types) are just as effective in the recovery of clinicallysignificant anaerobes as the more complex roll-tube and chamber methodsemploying prereduced media. Liquid media were disappointing as a "back-up"system but chopped-meat glucose was superior to two thioglycolate formula-tions. Growth of all anaerobes was poorer on selective media, but these mediawere very helpful in the workup of specimens containing mixed growth ofanaerobic and facultative organisms. A variety of different anaerobes wasisolated, but no very fastidious or extremely oxygen-sensitive organisms wererecovered. This suggests that such organisms may not play a significant role incausing clinical infections.

There has been an increased interest in therole of anaerobic bacteria in clinical infectionsin recent years. However, the true incidence ofanaerobic infections is unknown. Older studies(4, 7, 14) provided incidence figures of 2 to 10%,whereas a recent report (11) indicated recoveryof anaerobes from 85% of clinical specimens.These studies have all had serious deficienciesincluding inadequate methods of collection,transport, culture, and identification or a lackof clinical correlation with the culture results,or both.One of the major impediments to an expan-

sion of our knowledge about anaerobic infec-tions is the rather poor state of anaerobicbacteriology in most hospital microbiology lab-oratories. Many of these laboratories would liketo improve their anaerobic techniques, but areuncertain as to which methods are necessaryand are technically and economically feasible.While some workers have decried the inade-quacies of jar and liquid methods and recom-mended the more complex roll tubes and ana-erobic chamber, others have protested thatthese latter methods are too complex, timeconsuming, and expensive for routine use.The study which follows was designed to

determine which methods of anaerobic culture77

are adequate for the recovery of clinicallysignificant anaerobic bacteria. Precautionswere taken to obtain specimens free fromcontamination with normal flora (where ana-erobes abound) and to protect specimens fromoxygen by transporting them in anaerobic con-tainers. Close observation of patients by infec-tious disease physicians permitted an evalua-tion of the clinical significance of the anaerobesisolated. Each specimen was cultured usingliquid media, anaerobic jars, roll tubes, and ananaerobic chamber utilizing both selective andnonselective media. The recovery of anaerobesby these different methods was then compared.

MATERIALS AND METHODSClinical specimens. Specimens were obtained

from hospitalized patients of the Infectious DiseaseSection of Wadsworth Hospital Center. Each pa-tient, with one exception (see below), had a clinicalcourse consistent with an anaerobic infection. Allspecimens were obtained avoiding contaminationwith normal flora. Transtracheal aspiration (TTA)and thoracentesis were used for pulmonary speci-mens. Closed abscesses were aspirated using aneedle and syringe. Peritoneal fluid was aspiratedby transabdominal percutaneous needle punctureand tissue specimens were obtained using asepticsurgical techniques. As soon as the specimens were

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obtained, they were injected into a double-stopperedtransport tube containing oxygen-free CO2 or N2 (2).One milliliter of prereduced anaerobically sterilized(PRAS) peptone yeast glucose medium was includedin tubes used for transport of very viscous specimensor those with a volume of less than approximately 1ml. Tissue specimens were placed in the anaerobic"mini-jar" described by Attebery and Finegold (3).Transport containers were taken directly to theAnaerobic Microbiology Laboratory and immedi-ately processed or stored at 4 C usually for no longerthan 2 hr.

Processing of specimens. The transport tubewas placed in the anaerobic chamber where thespecimen was transferred, using a needle and syringe,to a second rubber-stoppered tube (appropriate forlater use with a gassing cannula). The chamber usedwas similar to that described by Aranki et al. (1).Anaerobiosis was monitored by use of methylene blueindicator strips (BBL, Division of Becton, Dickinsonand Co.). The chamber plated media were theninoculated using a standard 0.01-ml stainless-steelloop. This same inoculum was used for all plates andtubes included in this study. Plates were thenstreaked and placed inside a GasPak (BBL) anaero-bic jar which was sealed. Palladium-coated aluminapellet catalysts were included in the jar which, alongwith the remaining specimen, was then removedfrom the chamber. Our chamber had no heating unitand was not suitable for incubation purposes. Theroll tubes were then inoculated according to proce-dures described in the Outline of Clinical Methods inAnaerobic Bacteriology (V.P.I. Anaerobic Labora-tory, Blacksburg, Va. 2nd. revision, June, 1970). Thespecimen tube was continuously gassed out withoxygen-free CO, during the time when the stopperwas removed. Tube roller, roll-tube streaker, andgassing-out apparatus were constructed after thedesign described in the Outline.The remainder of the specimen was then used on

the open bench to inoculate plates for the GasPak jarand a jar made anaerobic by a system of evacuationof air and replacement with an oxygen-free gasmixture. The GasPak jars were set up by simplyadding 10 ml of water to the commercial packet(hydrogen-CO2 generator) and enclosing this in thejar which was then sealed. The evacuation-replace-ment jars were sealed and then a vacuum of 26 to 28inches of Hg was drawn. The jar was then filled withoxygen-free N., and the procedure was repeated fourtimes. The fifth fill was with a gas mixture contain-ing 80% N2, 10% H2, and 10% CO2. Each time ananaerobic jar was set up a "rejuvenated" catalystpacket was used. These palladium-coated aluminapellets were rejuvenated by heating to 160 C for 2 hr.A methylene blue indicator strip was enclosed ineach jar.The liquid media were the last to be inoculated.

The tubes of thioglycolate media were boiled for 5min before using, and the stopper was removed onlylong enough to allow inoculation. Plates for isolationof facultative organisms were inoculated with theremaining specimen. All containers were incubatedat 37 C.

Media. The following set of four plates was inocu-lated for both the GasPak and the evacuation-replacement anaerobic jars: a Brucella blood-agarplate containing menadione (BMB), a neomycinblood-agar plate (NEO), and a laked blood-agarplate containing vancomycin and kanamycin (LKV).The preparation of these media has been describedby Finegold (6). A fourth plate consisted of thestandard BMB medium to which 0.1% dithiothreitol(D'IT) and 0.1% cysteine were added. Moore (10) hasshown that the inclusion of DTI in blood-agar platesfacilitates recovery of the fastidious anaerobe Clos-tridium novyi, type B, in the anaerobic jar. Platemedia were stored by wrapping in mylar bags withthe top folded over and taped and refrigerated at 4 C.They were generally used within 1 week of prepara-tion.

Three kinds of liquid media were utilized. Com-merical (BBL 11260) "regular" fluid thioglycolatemedium (R-thio) was prepared according to themanufacturer's directions. A so-called "concentratedthioglycolate" (C-thio) was prepared by adding 29.5g of the commercial fluid thioglycolate medium and 1ml of a hemin solution to 750 ml (rather than theusual 1 liter) of distilled water. To each 9-ml tube ofthis medium was added 1 to 3 ml of sterile asciticfluid and 0.01 ml of menadione. PRAS chopped-meatglucose (CMG) medium was either purchased fromScott Laboratories (Chapel Hill, N. C.) or prepared inour laboratory. The PRAS CMG was not gassed outwith CO2 during the time the stopper was removedfor inoculation. In spite of this, less than optimumprocedure, the resazurin indicator did not becomeoxidized.PRAS media were prepared according to methods

in the V.P.I. Outline for CMG, roll tubes, and platedmedia used in the anaerobic chamber. The composi-tion of PRAS BMB, LKV, and NEO was similar tothat of the non-PRAS media except that sodium thio-glycolate and cysteine hydrochloride were includedas reducing agents. Roll tubes contained PRASbrain heart infusion agar (BHIA), BMB (utilizinglaked blood), LKV, and NEO (also utilizing lakedblood) media. These tubes were stored as agar deepsuntil ready for use at which time they were melteddown and, as required, laked blood, menadione, andvancomycin were added and mixed, and the tubeswere placed on the tube roller apparatus. PRASmedia for the anaerobic chamber (BMB, LKV, andNEO) were placed inside the chamber while still inthe heated liquid form where the plates were poured.After the plates had solidified, they were placed inanaerobic jars, removed from the chamber, andstored in the dark at room temperature. In the secondhalf of the study a home canning apparatus was usedto seal the plates inside gas-tight aluminum canswhich could then be refrigerated at 4 C.

Identification of anaerobes. Identification ofthe nonsporeforming gram-negative rods was bymethods outlined by Sutter and Finegold (15) in-cluding Gram stain and colonial morphology,susceptibility to certain antibiotic discs, growth inbile and deoxycholate, as well as the other testsindicated in the V.P.I. Outline. Other anaerobes were

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identified according to the V.P.I. methods includingthe gas chromatographic analysis of metabolicproducts. Most organisms were identified to genus

and species although a number could only be identi-fied to the genus level because of considerations oftime or inconclusive test results.

Evaluation of recovery of anaerobes. Cultureswere periodically examined for evidence of anaerobicgrowth. Jars were opened and the plates were in-spected, and jars were resealed according to methodsalready described. Roll tubes were inspected withoutremoving the stoppers, or, if picking of colonies was

required, the stoppers were removed and all proce-

dures were carried out while gassing the tubes withoxygen-free CO2. Liquid media were examined byGram stain and by subculturing to plated media.The jar containing anaerobic chamber plates was

placed in the chamber for inspection of plates. Finalsubcultures for all methods were made to plateswhich were incubated in GasPak jars. In no instancewas an organism which had been originally isolatedlost in the process of subculturing. In other words,once the organisms had grown up in the originalculture in the liquid or PRAS roll tube or chambermedia, they could be subcultured to plated media inroom air and incubated in the GasPak anaerobic jarwithout loss of viability.A semi-quantitative scheme was devised to evalu-

ate the growth of the anaerobes. For streaked plates a

1+ to 4+ score was used with 1+ representingcolonies present only in the primary streak area and4+ representing heavy growth in the third streakarea; 2+ and 3+ represented intermediate degrees ofgrowth. Colonial growth in streaked roll tubes was

also evaluated on a 1+ to 4+ scale, with 1+ repre-

senting colonies present only in the bottom one-

fourth of the tube and 4+ representing heavy growthin both the bottom half and top half of the tube; 2+and 3+ represented intermediate degrees of growth,primarily confined to the bottom half of the tube.Any growth in liquid media was considered "posi-tive," and the degree of growth was not graded.Because this is only a semi-quantitative method andthe grading was necessarily different for plates androll tubes, a difference in growth of at least 2 grades,i.e., from 1+ to 3+ or 2+ to 4+, was required beforethe difference in recovery of organisms was consid-ered significant. Plates and tubes were examined at24, 48, and 72 hr, and the presence or absence andquantitation of growth were recorded. Subculturesfor isolation and identification were made after 72 hr.The original plates and tubes were observed atirregular intervals over the next 2 weeks for theappearance of new anaerobic growth. Anaerobicisolates which could not be identified soon afterisolation were stocked for future identification bysubculturing into a skimmed milk medium andfrozen at -65 C.

RESULTS

Specimens. A total of 23 specimens were

included in the study. There were seven pulmo-nary specimens obtained by TTA. There were

four intraabdominal abscesses including twosubphrenic abscesses, a liver abscess, and anabscess adjacent to the bowel. Four otherabscesses included a brain abscess, an abscessof the retroperitoneal space, an abscess of thechest wall, and a lung abscess which wassurgically drained. There were three specimensof peritoneal fluid obtained by transabdominalneedle puncture. Five other specimens inc-luded an aspiration of a sinus tract leading to asite of osteomyelitis, pus from an empyemacavity drained surgically, a surgically obtainedtissue specimen (soft tissue infection and os-teomyelitis involving the foot), an aspiration ofthe gallbladder, and an aspiration of materialfrom an area of cellulitis and gangrene of a toe.Of the total of 23 specimens, 9 were frominfections in the respiratory tract and another 9were from infections directly related to theintestinal tract or hepatobiliary system. Be-cause our hospital is primarily composed ofmale veterans, we processed no specimens fromthe female genital tract which is a not uncom-mon site of anaerobic infection.Anaerobic culture data. A single anaerobe

was recovered in pure culture in four speci-mens. Multiple anaerobes were found in asingle specimen. A single anaerobe and oneor more facultative organisms were recoveredfrom four specimens and multiple anaerobesand facultative organisms were recovered from14 others. Five of the 23 specimens containedonly anaerobes and 18 contained mixtures ofanaerobes and facultative organisms. Thirteenof these 18 specimens contained multiple facul-tative organisms.Table 1 lists the 51 different isolates of

anaerobic bacteria recovered from the 23 speci-mens. Twenty-seven of these were non-sporeforming gram-negative rods; includedwere 12 isolates of Bacteroides fragilis, 10isolates of B. melaninogenicus, 4 isolates ofFusobacterium nucleatum, and 1 isolate of F.necrophorum. There were four isolates belong-ing to the genus Clostridium, including oneisolate each of C. perfringens, C. ramosum, C.subterminale, and a Clostridium which was notspeciated. There were six isolates of the genusPropionibacerium, and three of these wereidentified as P. acnes whereas three others werenot speciated. Three members of the genusEubacterium were recovered but were not spe-ciated. Eleven anaerobic cocci were isolatedincluding two so-called "microaerophilic strep-tococci." These organisms would not grow onplates incubated in room air but grew onanaerobic plates and in the presence of 2 to 5%CO2. There were three isolates of Peptococcus

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asaccharolyticus. The four Peptostreptococcusisolates included two P. magnus, one P. inter-medius, and one P. anaerobius. An additionalgram-positive organism could be identified nofurther than "anaerobic coccus." There wasone isolate of a nonspeciated Veillonella. Inaddition to the 51 isolates included in thisstudy, we have also recovered isolates of B.oralis, B. corrodens, and P. prevotii from clini-cal specimens by the GasPak method.Comparative data. Tables 2 to 4 illustrate

the comparative recovery of the anaerobes bythe various isolation methods. The three differ-ent liquid media are presented here as separate"methods." Growth in any of the media used ina given method was considered evidence ofrecovery by that method.Table 2 shows that the R-thio and C-thio

TABLE 1. Recovery of specific anaerobesa

No. of isolatesAnaerobesreordrecovered

Bacteroides ...................... 22B. fragilis ...................... 12B. melaninogenicus ............. 10

Fusobacterium ............... 5F. nucleatum ................... 4F. necrophorum ................ 1

Clostridium ............... 4C. perfringens .................. 1C. species ........................ 3

Propionibacterium.............. ..6Eubacterium ............... 3Peptococcus ............... 3Peptostreptococcus ............... 4"Anaerobic coccus"................. 1Veillonella ............... 1"Microaerophilic Streptococcus" 2

Total ........................ 51

a Number of different isolates recovered, consider-ing all isolation methods; total = 51.

liquid media demonstrated consistently poorerrecovery of B. fragilis, B. melaninogenicus, andF. nucleatum than did the other methods,including the CMG. The R-thio and C-thiorecovered 60 to 75% of these isolates comparedto a 78 to 100% recovery rate by other methods.In addition, the roll tubes and plates demon-strated no consistent differences in yield oforganisms, using the semi-quantitative gradingsystem.Table 3 provides data on the recovery of

gram-positive nonsporeforming rods and Clos-tridium. The liquid media failed to grow oneisolate of P. acnes and one isolate of C.ramosum. In addition, the roll tubes failed togrow one isolate of C. perfringens and one

isolate of C. ramosum for a recovery rate of50%for Clostridium. Recovery rates, with the aboveexceptions, were 100%, and there were no

consistent differences in yield by semi-quan-titative grading.The data for the anaerobic cocci is given in

Table 4. Recovery was 100% by each methodwith the following exceptions: the liquid mediaand roll tubes failed to grow two isolates ofPeptostreptococcus (P. intermedius and P.anaerobius) and one isolate of P.asaccharolyticus for recovery rates of 50% (33%for CMG) and 67%, respectively. The semi-quantitative grading system revealed no differ-ences in yield by the different methods.The liquid media were disappointing as a

"backup" method. There were only two in-stances in which isolates not recovered by othermethods grew in liquid media. On the otherhand, there were eight isolates recovered by atleast one of the other methods which were notisolated from liquid media.Table 5 provides data on the recovery of all

anaerobic isolates by different media. Data forthe GasPak jar and the evacuation-replace-

TABLE 2. Comparative recovery of anaerobic nonsporeforming gram-negative rods

No. of GasPak Evacuation- Roll LiquidaAnaerobes iso- replacement Chamber

lates jar tubes CMG R-thio C-thio

Bacteroidesfragilis 12" 10c (83)d 11 (92) 10(83) 10 (83) 10 (83) 9(75) 9 (75)B. melaninogenicus 10 9 (90) 9(90) 8(80) 7/9e (78) 8/9e (89) 7 (70) 6(60)Fusobacterium nucleatum 4 4 (100) 4 (100) 4 (100) 4 (100) 4 (100) 3 (75) 3 (75)F. necrophorum 1 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100)

a CMG, chopped-meat glucose; R-thio, regular commercial thioglycolate; C-thio, concentrated thioglyco-late.

b Total number of different isolates recovered considering all methods.c Number of isolates recovered by each method.dPer cent recovery.' Indicates this method not done with one specimen.

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TABLE 3. Comparative recovery of anaerobic nonsporeforming gram-positive rods and Clostridium

No. of GasPak Evacuation- Roll LiquidaAnaerobes ioolat jar replacement tube Chamber M Rthio C-thi

jarCM Rtho Cti

Propionibacterium 6b 6C (100)d 6 (100) 6 (100) 5/5' (100) 4/5' (80) 5' (83) 5' (83)Eubacterium 3 3 (100) 3 (100) 3 (100) 3 (100) 3 (100) 3 (100) 3 (100)Clostridium 4 4 (100) 4 (100) 2 (50) 3/3e (100) 3' (75) 3' (75) 3' (75)

aCMG, chopped-meat glucose; R-thio, regular commercial thioglycolate; C-thio, concentrated thioglyco-late.

"Total number of different isolates recovered considering all methods.c Number of isolates recovered by each method.d Per cent recovery.'Indicates this method not done with one specimen.' Same isolate not grown by each of these methods.

TABLE 4. Comparative recovery of anaerobic cocci

No. of GasPak Evacuation- Roll Liquid"Anaerobes | isolates | jar replacement tube Chamberisolatesjar .ja tueCMG R-thio C-thio

Peptostreptococcus 4b 4C (100)d 4 (100) 2 (50) 4 (100) 1/3e f (33) 2' (50) 2' (50)Peptococcus 3 3 (100) 3 (100) 2 (67) 3 (100) 2' (67) 2' (67) 2' (67)"Anaerobic coccus" 1 1 1 1 1 1 1 1Veillonella 1 1 1 1 1 1 1 1"Microaerophilic 2 2 2 2 2 2 2 2

Streptococcus"

a CMG, chopped-meat glucose; R-thio, regular commercial thioglycolate; C-thio, concentrated thioglyco-late.

° Total number of different isolates recovered considering all methods.c Number of isolates recovered by each method.d Per cent recovery.' Same isolate not grown by each of these methods.' Indicates this method not done with one specimen.

ment jar are grouped together under "Ana-erobic Jars" since the same media were used inboth. Growth in one jar but not the other isrepresented by fractional numbers, i.e., 7.5,8.5, etc. The growth of B. melaninogenicus wasconsistently poorer on selective media (LKVand NEO) with recovery rates of 40 to 78%compared to 71 to 90% on nonselective media.The same situation existed with B. fragilis andLKV media with rates of 46 to 63% compared to71 to 83% for the other media.

Isolates of Eubacterium did not grow onNEO and Clostridium did not grow on LKV. Inaddition, the recovery of all the gram-positiverods was consistently poorer on the selectivemedia (LKV and NEO) than on other media(rates of 0 to 50% compared to 50 to 100%).

Colony size was not noticeably larger nor didcolonies appear earlier on one type of mediumor in one specific method than in any of theothers. The possible exception is DTT wherecolonies of many different organisms appearedslightly larger than on other media. However,

this size difference was not considered signifi-cant and growth on DTT was not superior bysemi-quantitative grading or earlier appear-ance of colonies.Although recovery of most anaerobes was

somewhat poorer on the selective media (LKVand NEO), these media were considered veryuseful. In particular, specimens containingheavy growth of facultative organisms weremore easily evaluated using selective mediawhich inhibited the growth of most of theseorganisms. The inclusion of laked blood in theLKV medium greatly enhanced the appearanceof the pigment of B. melaninogenicus, makingcolonies of this organism easily recognizable.The combined use of selective and nonselectivemedia was very helpful in the isolation of allorganisms, including those inhibited on LKVand NEO.

Roll tubes were technically more difficultand more time consuming to work with thanthe other methods. Colonies were often not wellseparated and difficult to pick cleanly with an

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METHODS FOR ANAEROBIC BACTERIA ISOLATION

inoculating wire. Specimens containing heavygrowth of facultatives were especially trou-blesome, necessitating extensive subculturingfor isolation of individual colony types. Selec-tive media were less helpful in roll tubes thanwith the other methods. Inclusion of blood(even laked blood) in roll tube media madecolony types less recognizable. Different colonytypes were more difficult to recognize and lessdistinctive, even with the use of a dissectingmicroscope. The characteristic colonial mor-

phology described for many anaerobes was notapparent on examination of the roll tubes. Thelarger diameter (25 mm) tubes were used in thelatter part of the study and were preferable interms of colony separation and recognition, butrecovery rates did not improve.

DISCUSSIONSeveral authors have emphasized that older

methods of anaerobic culture, in particular theanaerobic jar and liquid media containingreducing agents, are inadequate for the recov-ery of many important anaerobes. Moore et al.(11) reported the recovery of 144 anaerobicisolates from 81 clinical specimens using theroll-tube method. Moreover, in their experi-ence over 85% of about 300 clinical specimenshave contained obligate anaerobes. However,the data of Moore et al. provide no assurancesthat their specimens came from patients withclinically significant infections or were col-lected so as to avoid contamination with nor-mal flora. Since their study was not a compara-tive one, their data cannot be accepted as adefinitive demonstration of the superiority ofroll tubes for the isolation of clinically signifi-cant anaerobes.Vargo et al. (Bacteriol. Proc., p. 109, 1971)

have done a comparative study showing signifi-cantly better recovery of anaerobes in an an-

aerobic research laboratory (using PRAS mediaand the anaerobic chamber or jars) comparedto that in a routine clinical laboratory. Howe-ver, in this study there were a great manyvariables other than the culture method whichmay have contributed to the different yield ofanaerobes.McMinn and Crawford (8) reported that the

combined use of thioglycolate and a GasPak jarrecovered only 28.6% of anaerobes isolated bythe roll-tube method. However, their datasuggest that a number of their specimens (oralcavity, sputum, ulcers, drain sites, urine) musthave been contaminated with normal flora.Furthermore, as the authors state, "Most spec-

imens were submitted aerobically."

Spears and Freter (13), Drasar (5), andAranki et al. (1) have all demonstrated thesuperiority of the use of PRAS media in rolltubes or the anaerobic chamber for recovery ofanaerobes over conventional jar methods.However, these studies dealt with normal in-testinal flora, not clinical specimens.The data presented in the present study

suggest conclusions somewhat different fromthose of the above-mentioned reports. Our dataindicate that, when clinical specimens areobtained avoiding contamination with normalflora, are immediately placed under anaerobicconditions, and are transported in an anaerobiccontainer, the recovery of anaerobes with thejar method is as good as with PRAS media inroll tubes or on plates in the anaerobic cham-ber. Criticism of the anaerobic jar method isbased primarily on exposure of the specimen tooxygen during inoculation and the oxidizedstate of the media. Additional time is requiredbefore the atmosphere in the jar becomesreduced. However, apparently most (and per-haps all) clinically significant anaerobes cansurvive and grow under these conditions, prov-iding the specimen is placed in an anaerobicatmosphere as soon as it is obtained, i.e.,through the use of anaerobic transport contain-ers.

Since each of our patients was followed bythe Infectious Disease Section, we were able toinsure that the anaerobic isolates included inthis study came from clinically significantinfections. A single exception was a pure cul-ture of Propionibacterium spp. recovered froma TTA. This patient's course suggested his lungdisease had a noninfectious etiology. Pro-pionibacterium was also recovered from fiveother specimens but in mixed culture withother anaerobes and (in four instances) faculta-tive organisms. These organisms are part of thenormal skin flora, and their role in causingdisease has not been adequately determined.However, several authors (11, 12; R. F. Betts,H. Short, and V. R. Dowell, 11th Intersci. Conf.Antimicrob. Ag. Chemother. Proc., p. 71, 1971)have reported experiences suggesting a patho-genic role for Propionibacterium. Our data donot allow us to evaluate the specific role, if any,of our Propionibacterium isolates in the infec-tions with which they were associated.The anaerobic jar is unquestionably not

suitable for study of normal flora which con-tains organisms more fastidious in their re-quirements for anaerobiosis and others that areextremely sensitive to oxygen. These so-calledextremely oxygen-sensitive anaerobes (H. R.

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Attebery, L. J. Nastro, and S. M. Finegold,Bacteriol. Proc., p. 108, 1971) will die if ex-posed to room air for more than 5 min. On thebasis of our limited data, it appears that thefastidious anaerobes of the intestinal and oralflora may not be significant causes of clinicalinfections since we recovered no anaerobes, bythe chamber or roll-tube methods, that did notgrow in the anaerobic jars.The results of this study should have consid-

erable importance for the clinical laboratory.Adequate anaerobic work can be performedusing conventional plates and the anaerobic jaras long as proper collection and transport ofspecimens is carried out. Strong efforts shouldbe directed towards overcoming deficiencies inthe latter two areas. The roll-tube and chambermethods which are time consuming, techni-cally difficult, and require PRAS media andspecialized equipment apparently are not nec-essary for isolation of anaerobes in the clinicallaboratory. The technical complexities of work-ing with roll tubes, in fact, were probablyresponsible for the slightly poorer recoveryrates in our study, rather than any inherentdefect in the method itself. In spite of the factthat adequate clinical anaerobic bacteriologycan be carried out with jars, the chamber androll-tube methods may be advantageous forlaboratories processing large numbers of speci-mens. Individual roll tubes and plates insidethe chamber can be examined without disturb-ing the anaerobic atmosphere.Although CMG performed better than the

other liquid media, the overall recovery ofanaerobes in liquid media was disappointing.There were eight instances in which anaerobeswhich did not grow in liquid media wererecovered by other methods, whereas therewere only two instances where the reverse wastrue. Suboptimal use of CMG may have con-tributed to this poor record. CMG is a PRASmedium, but we removed the stopper for inocu-lation without gassing the tube with oxygen-free CO2 or N2. We were attempting to simu-late conditions in the average hospital labora-tory which would not have a gassing apparatus.However, even brief exposure of PRAS mediato oxygen can cause oxidation of the reducingagents (even though our indicators did notbecome oxidized) leading to a poorer anaerobicenvironment. The current availability ofPRASCMG in anaerobic vials with rubber dia-phragm stoppers (Hyland Division, TravenolLaboratories Inc., Costa Mesa, Calif.) shouldeliminate this problem. The specimen can beinoculated into the CMG vial, and subculturescan be carried out with a needle and syringe

without exposure of either the specimen ormedium to oxygen.The use of selective media (LKV and NEO)

in this study was of great benefit in theisolation of anaerobes in the presence of heavygrowth of facultative organisms. Considerabletime and effort was saved. However, since thereis a decrease in recovery of most anaerobes onselective media, a suitably enriched nonselec-tive medium should always be inoculated alongwith the selective medium.Based on the results of this study, our

current recommendation for adequate proces-sing of clinical specimens by a hospital mi-crobiology laboratory would include the follow-ing: (i) emphasis placed on proper collection ofspecimens for anaerobic culture avoiding con-tamination with normal flora; (ii) provision fortransport of specimens in a container with anoxygen-free atmosphere; (iii) immediate plat-ing of specimens once they are removed fromthe anaerobic transporter and immediateplacement of plates in a jar which is immedi-ately set up for anaerobic conditions; (iv) use ofone plate of enriched medium, such as BMB,and at least one plate of a selective medium,such as LKV, as well as a tube of PRAS CMGliquid for each specimen. An acceptable alter-native to the immediate setting up of anaerobicjars as soon as a specimen is plated would bethe storage of inoculated plates in a CO2-gassedjar, such as that described by Martin (9), untilenough plates have accumulated to fill and sealajar.

LITERATURE CITED

1. Aranki, A., S. A. Syed, E. B. Kenney, and R. Freter.1969. Isolation of anaerobic bacteria from humangingiva and mouse cecum by means of a simplifiedglove box procedure. Appl. Microbiol. 17:568-576.

2. Attebery, H. R., and S. M. Finegold. 1969. Combinedscrew-cap and rubber-stopper closure for Hungatetubes (pre-reduced anaerobically sterilized roll tubesand liquid media). Appl. Microbiol. 18:558-561.

3. Attebery, H. R., and S. M. Finegold. 1970. A miniatureanaerobic jar for tissue transport or for cultivation ofanaerobes. Amer. J. Clin. Pathol. 53:383-388.

4. Bornstein, D. L., A. N. Weinberg, M. N. Swartz, and L.J. Kunz. 1964. Anaerobic infections-review of currentexperience. Medicine 43:207-232.

5. Drasar, B. S. 1967. Cultivation of anaerobic intestinalbacteria. J. Pathol. Bacteriol. 94:417-427.

6. Finegold, S. M. 1970. Isolation of anaerobic bacteria, p.265-279. In J. E. Blair, E. H. Lennette, and J. P.Truant (eds.), Manual of clinical microbiology.American Society for Microbiology, Bethesda, Md.

7. Goldsand, C., and A. I. Braude. 1966. Anaerobic infec-tions. Disease-a-Month. Nov., 1966, 62 pp.

8. McMinn, M. T. and J. J. Crawford. 1970. Recovery ofanaerobic microorganisms from clinical specimens inprereduced media versus recovery by routine clinicallaboratory methods. Appl. Microbiol. 19:207-213.

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METHODS FOR ANAEROBIC BACTERIA ISOLATION

9. Martin, W. J. 1971. Practical method for isolation ofanaerobic bacteria in the clinical laboratory. Appl.Microbiol. 22:1168-1171.

10. Moore, W. B. 1968. Solidified media suitable for thecultivation of Clostridium novyi type B. J. Gen.Microbiol. 53:415-423.

11. Moore, W. E. C., E. P. Cato, and E. V. Holdeman. 1969.Anaerobic bacteria of the gastrointestinal flora andtheir occurrence in clinical infections. J. Infect. Dis.119:641-649.

12. Smith, L. D. S., and L. V. Holdeman. 1968. Propionibac-teria and anaerobic Corynebacteria, p. 138-146. In

The pathogenic anaerobic bacteria. Charles C Thomas,Springfield, Ill.

13. Spears, R., and R. Freter. 1967. Improved isolation ofanaerobic bacteria from the mouse cecum by main-taining continuous strict anaerobiosis. Proc. Soc. Exp.Biol. Med. 124:903-909.

14. Stokes, E. J. 1958. Anaerobes in routine diagnosticcultures. Lancet. 1:668-670.

15. Sutter, V. L. and S. M. Finegold. 1971. Antibiotic discsusceptibility tests for rapid presumptive identifica-tion of gram-negative anaerobic bacilli. Appl. Mi-crobiol. 21:13-20.

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