APPLIED MICROBIOLOGY, May 1974, p. 961-979 Vol. 27, No. 5Copyright 0 1974 American Society for Microbiology Printed in U.S.A.

Human Fecal Flora: The Normal Flora of 20Japanese-Hawaiians

W. E. C. MOORE AND LILLIAN V. HOLDEMANAnaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

Received for publication 21 January 1974

Quantitative and qualitative examination of the fecal flora of 20 clinicallyhealthy Japanese-Hawaiian males was carried out by using anaerobic tubeculture techniques. Cultural counts were 93% of the microscopic clump counts.Isolated colonies were selected in a randomized manner to give an unbiased sam-pling of the viable bacterial types. Each isolate was characterized for speciesidentification. From a total of 1,147 isolates, 113 distinct types of organisms wereobserved. Statistical estimates indicate that these types account for 94% of theviable cells in the feces. The quantitative composition of the flora of this group ofpeople, together with differential characteristics of previously unreported spe-cies, is presented for those kinds of bacteria which each represented at least0.05% of the flora.

As a result of suggestions by workers at theWright-Fleming Institute of possible relation-ships between the bacteria of the human colonand the incidence of colon or other cancer (2-4,35-37), there has been increased interest in thedistribution of different kinds of bacteria infeces.

Several hundred species have been reportedfrom the fecal flora since Escherich (22) firstdescribed "Bacterium coli commune," andthere have been innumerable reports of thedistribution of various morphological or culturalbacterial groups (1, 5-9, 13, 15-20, 23-28, 30-34, 42-48, 50-62). Still, many questions remainconcerning the identity and frequency of indi-vidual bacterial species in the flora, the varia-tion in their relative numbers in different speci-mens or subjects, and the factors which maychange the composition of the flora that cannow be cultured.Van Houte and Gibbons (58) reported micro-

scopic counts of 3.2 x 1011 bacterial cells per g(wet weight) of feces. This would be about 1012to 1.6 x 1012 per g (dry weight). Since theaverage bacterial cell of the fecal populationoccupies about 1 Am3, the cells occupy morethan 30% of the total wet volume. In the lastdecade, it has been demonstrated repeatedlythat more than 50% of the human fecal flora iscultivable if strictly anaerobic techniques simi-lar to those developed by Hungate (40) areemployed (21, 49).The present work is an effort to describe and

quantitate those presently cultivable species,

each of which comprises at least 0.05% of thenormal fecal flora of this human population.These data are to be used subsequently forcomparisons with flora of other groups of peopleand to evaluate conditions that may affect therelative concentrations of bacterial specieswhich are numerically most significant.

MATERIALS AND METHODSFeces from 20 clinically healthy Japanese-

Hawaiian males, ages 60 to 80, whose normal dietsrange from Oriental to Western, were studied. Theseindividuals were in an epidemiological health studyconducted by the National Cancer Institute of theNational Institutes of Health. The regular weekly dietof most individuals included western foods, such asbeef, bacon, preserved meats (Spam, wieners, etc.),fresh vegetables (lettuce, tomatoes, corn), and Orien-tal foods, such as rice, fish, tofu, and Oriental-stylevegetables. Ten of the 20 ate raw fish; 13 drank milk;18 ate eggs; and 11 of 12 questioned ate chicken.

Previous work in this laboratory on specimens fromNorth Americans indicated that different methods forshipping feces produced changes in the compositionand decreases in the statistical "coverage" values (29)of the viable flora. Methods tested included freezingin liquid nitrogen or dry ice and alcohol, with orwithout glycerol diluent (14). Storage or shipping atambient temperature (20 to 30 C) under CO, for 2days produced the least change of any method tested,with no statistical difference from results obtainedwith immediate culture. However, to eliminate anypossible changes in fecal specimens during shipment,all specimens were cultured immediately after collec-tion.

Preliminary trials indicated that different 1-g sub-samples of the stool often differed in bacterial compo-

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sition. Therefore, each entire stool was mixed toobtain an estimate of the bacterial population. Theentire fecal specimen was collected in a plastic bagand was immediately transported to the laboratory.Processing was initiated within 5 min of defecation.The bag was flushed with 0,-free CO,, and the stoolwas thoroughly mixed by kneading. Approximately 1g of the mixed feces was transferred to 9 ml ofprereduced dilution salts solution (38) under O,-freeCO,. Actual sample weight was determined from theincreased weight of the first dilution tube. Aftervigorous shaking to completely disperse the sample,serial 10-fold dilutions were prepared with vigorousshaking of each restoppered tube before subsampling.

Direct microscopic clump counts were made fromduplicate smears of 0.01 ml of the 10' dilution spreadover 1 cm2. Smears were heat-fixed and gently Gram-stained. Six edge fields and four center fields werecounted by two people on each of the two squares.Both microscopic and cultural counts were correlatedfor the actual sample size.

Duplicate rumen fluid-glucose-cellobiose agar(RGCA) roll tubes (25 x 142 mm) were inoculatedunder 0,-free CO, with 1 ml each of 10', 10', and 1010dilutions and incubated at 37 C. After incubation for16 to 72 h, the tubes were transported in an uprightposition from Honolulu, Hawaii, to Blacksburg, Va.Transport time was 24 h. After a total of 5 days ofincubation at 37 C, roll tubes were marked with spirallines, and the colonies were counted by at least twoindividuals. Counts between 30 and 300 colonies pertube were used to estimate the cultural count.

Moisture content of the fecal specimens was deter-mined on 10-g samples of each stool (in duplicate foreach specimen of sufficient size). Samples for thisdetermination, and for chemical analyses to be re-ported elsewhere, were quick frozen in sealed plasticvials in liquid nitrogen and kept frozen until ana-lyzed. Weighed samples were lyophilized for 6 h andreweighed to determine percent dry matter by differ-ence. Direct microscopic clump counts and culturalcounts were then corrected to a dry weight basis.

In an attempt to determine the ratio of differentkinds of organisms in the normal flora, every wellisolated colony, regardless of appearance, was pickedin succession (top to bottom of the tube) until 55colonies had been picked. Colonies that were so closetogether that they could not be picked separately orthat were under an obvious area of surface spreadingwere not picked. The colonies were observed under adissecting microscope (10 to 15 x) as they werepicked, and the roll tubes were flushed with 0.-freeCO, during the picking procedure by using a CO,cannula and picking needle guide attached to thedissecting microscope stage (Bellco Glass Co., Vine-land, N.J.). Colonies were picked into 3 ml of Sweet Ebroth (38) because, in studies not included here, itwas found that many of the isolates required one oranother disaccharide for growth.

All cultures resulting from the original single colo-nies were streaked on supplemented brain heart infu-sion agar (BHIA) or Sweet E agar streak tubes andagain picked into 3 ml of Sweet E broth. If more than

one colony type or more than one morphotype was ob-served, an attempt was made to isolate and charac-terize each. Each isolate was characterized accordingto procedures described previously (38). For mostisolates, about 40 different media were inoculatedand analyzed. In addition to usual biochemical testsand analyses for metabolic alcohols and acids, manyisolates were tested for their ability to produce hydro-gen. This characteristic was found to be useful fordifferentiating species with otherwise similar reac-tions. To detect hydrogen, 1 ml of head gas (usuallyfrom a fructose broth culture), taken with a needlethrough the edge of the rubber stopper, was chro-matogrammed on a 6-ft by 316 in (182.88 cm by 0.48cm), 80 to 100-mesh silicic acid column at 40 C with 16ml of N, carrier gas per minute, using a thermalconductivity detector. Relative amounts of hydrogenwere scored according to peak height. "Four plus"values represent more than 2% hydrogen in the headgas. Hydrogen was produced by many strains that didnot produce obvious gas in deep agar tubes.

Isolates were identified according to criteria pub-lished in the Anaerobe Laboratory Manual (38),companion manuscripts (11, 12, 39), and as discussedbelow.The data have been analyzed statistically to deter-

mine the composition of the flora of these people as apopulation and therefore do not consider person-to-person variation and day-to-day variation withinpersons. These variables will be discussed in a laterpublication.

RESULTS AND DISCUSSION

Microscopic and cultural counts. The mi-croscopic and cultural counts obtained on the20 specimens are given in Table 1. The micro-scopic count we obtained is not as reproducibleas the cultural count. For the microscopic esti-mates, we used stained slides rather than acounting chamber, because the prepared slideswere shipped from Honolulu to Blacksburg forcounting. Our values for direct microscopiccounts are generally lower than those reportedby van Houte and Gibbons (58). This may re-sult from loss of cells from the slide duringstaining, even though care was used to mini-mize losses by very gentle rinsing and Gram'sstaining procedures, or because we report aclump count rather than a single cell count.Pairs and short chains of cells that appeared tobe firmly attached to each other and might beexpected to give rise to a single colony werecounted as one, as in the standard methods forexamination of dairy products. Van Houte andGibbons counted cells in a Petroff-Haussercounting chamber with simple stain, whichmight produce slightly high values because ofthe difficulty of distinguishing organisms fromstained debris. The same error can occur onstained slides, but may be decreased when a

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decolorization step is included. Van Houte andGibbons' counts, averaging 31.6 x 1010 per g(wet weight) may be a better estimate than ours,which averaged 11.2 x 1010 per g (wet weight).However, we have found that the average mois-ture content of Japanese-Hawaiian feces ex-ceeds that of North American feces. The mois-ture content of specimens examined by the twolaboratories might account for nearly all of thedifference observed in the total microscopiccounts. The dry matter content of individualspecimens in the present study had nearly a

threefold variation, as shown in Table 1. If weassume that the count of van Houte and Gib-bons is based on specimens of equal moisturecontent, and upon clump count rather thansingle cell count, 31.3% of their average micro-scopic count was cultured in the present study,whereas 87.96% of our microscopic count (pergram, wet weight) was cultured. The true cul-tural recovery probably falls within these ex-tremes.Comparison of van Houte and Gibbons' cul-

tural counts with those obtained here indicatesthat strict anaerobic techniques and specialmedia are essential. By using blood agar platesand anaerobe jars, they obtained an averagecultural count of 8.3 x 10' colonies per g (wetweight). With RGCA in pre-reduced roll tubes,we obtained an average cultural count of 9.9 x1010 on a wet weight basis.

Reports of counts corrected for moisture as

given by Attebery et al. (5) may provide a betterbasis for comparing results. Cultural counts inthe present study averaged 1011167 (4.75 x1011)/g dry matter, 93.87% of the average micro-scopic count/g dry matter. This compares veryfavorably with an average on normal specimensof 1011.36 (2.27 x 10'1)/g dry matter obtained byAttebery et al. (5) by using glove box proce-dures.Except for specimen C-3,,95.4% of the total

original "colonies" picked were characterized.Because recovery was poor from the first 55"colonies" picked from specimen C-3, coloniesfrom additional tubes were picked after 7 daysof incubation. In all, only 44 viable subcultureswere recovered from the 100 colonies pickedfrom C-3. This fecal specimen had a moisturecontent of 87.5% and the appearance of diar-rhea. However, the subject stated that this stoolwas of normal consistency for him. There were

no unusual problems with cultural recoveryfrom the other 19 specimens, some of which alsohad high moisture levels.With the exception of specimen C-3, only

occasional original colonies did not grow on

TABLE 1. Microscopic and cultural counts from 20fecal specimensa

Counts/g of drySubject0 Dry matter matterc

DMCCd Cultural

1 31.6 26.2 35.22 25.8 32.0 47.63 12.5 38.0 45.66 34.2 23.5e7 49.1 56.0e8 31.2 28.5 22.59 20.0 39.1 42.510 16.5 18.1 52.911 20.2 130.1 58.812 22.1 142.8 60.213 23.6 117.5 43.114 32.2 30.1 20.315 35.1 80.9 49.716 16.8 44.3 70.117 12.4 47.3 54.318 19.9 88.5 136.419 15.4 22.0 44.221 14.6 17.6 18.022 24.0 18.7 37.323 12.6 6.9 32.2

Average 21.5 50.6 47.5Standard deviation 39.7 25.4

aPaired t test between microscopic and culturalcounts, t = 0.38 (19 df = ns).

b Samples 4 and 5 were duplicate specimens used inshipping studies; specimen 20 was from a differenttreatment population. Therefore, they are not in-cluded in this study.

c Figures shown to be multiplied times 100.d DMCC, Direct microscopic clump count.'The estimate is based on 21.5% dry matter (the

average for this population). There was insufficientsample to make a direct determination of the percent-age of moisture.

subculture in Sweet E. Many of these "colo-nies" were originally described as "unusual" inappearance or "less than 0.2 mm in diameter."Some may have been unmelted bits of agar inthe original roll tube or particles of debris in therumen fluid of the RGCA. Some probably werebacterial types that we could not subculture.Also, because many isolates were obtained atthe same time, it was sometimes necessary tostore some of them. Isolates were lyophilizedand/or frozen at - 80 C and held at roomtemperature in Sweet E and chopped meat; afew of these did not survive any of these storageconditions.Kinds of bacteria detected. The species and

subspecies (kinds) of bacteria detected in indi-vidual fecal samples are given in Table 2.According to the formula of Good (29), the kinds

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of organisms isolated (based on 44 to 69 isolatesfrom individual specimens) account for 71 to95% of the total viable cells (71 to 95% "cover-age") in the individual fecal samples. The "%coverage" according to Good (29) = 1 - (num-ber of kinds observed once/total number exam-ined) x 100. The coverage of individual fecalspecimens can be calculated from the observeddata reported in Table 2 (e.g., for specimen C-1,% coverage = 1 - 18 [number of kinds seenonceJ/62 [number of isolates] x 100, which is71%.)There were 113 different kinds of organisms

detected among the 1,147 isolates examinedfrom the 20 people. In Table 3, the kinds oforganisms are listed in order of frequency oftheir occurrence in this human population. The113 kinds account for 94% of the viable cells (a94% coverage) of the fecal flora of these individ-uals as a population. For this calculation ofcoverage and for the calculated concentrationsof individual species, we used hypothesis H, ofGood (29), in which the expected frequency isdetermined by Sir (r + 1), where S = thenumber of kinds observed (113) and r = theobserved frequencies of the various kinds. Thesecalculations give more conservative estimates ofthe coverage and of the concentrations of indi-vidual species than would be obtained on thebasis of the observed values.The expected values obtained with hypothe-

sis H, and the observed values match quite wellfor those kinds that were observed 10 or moretimes. However, the statistical estimates indi-cate that the number of species seen only onceor twice is smaller than would be expected. Onerepresentative was observed for each of 38species, but hypothesis H., which fits the rest ofthe data well, predicts that 56.5 kinds should beobserved once. Thus, there is an indication thatwe may have combined in single groups somekinds which should actually be separated. Al-though characterization was fairly extensive,this appears to be inevitable with presentmethods of phenotypic characterization. Eventhough phenotypic differences usually indicatedifferent genetic groups, there are even moregenetically distinct organisms than now can bedifferentiated phenotypically. For example,Johnson (41) reported on the basis of DNA/DNA homology by competition experimentsthat there are at least three distinctly differentgenetic groups of organisms included in thosethat we label Bacteroides fragilis ss. thetaiotao-micron, two in ss. ovatus, and two in ss. fra-gilis. The genetic differences imply that thereare major metabolic or chemical differences

within the species or subspecies that now ap-pear to be phenotypically uniform. Attemptsto find phenotypic characteristics that differ-entiate the distinct genetic groups have not yetbeen successful.

Preliminary statistical analyses of these andadditional data by I. J. Good (personal com-munication) indicate that the total number ofdifferent kinds of bacteria in the intestinal tractat any one time probably exceeds 400 or 500species, but most of these are represented byless than 10' cells per g of feces (less than1/1,000 of the bacterial population).We recognize that the isolation methods used

would not have detected some organisms withvery specialized nutritional requirements ifthey, too, occurred in high numbers. However,it is reasonably certain that the isolationmethods used here did not select against thefacultatively anaerobic organisms in the fecalspecimens: (i) because the cultural recoveryaccounts for such a large proportion of the totalcellular population; (ii) because facultative spe-cies were isolated in numbers equal to or ex-ceeding those reported from aerobic culture (13,17, 23, 25-27, 31, 33, 34, 43, 45, 55-58, 62); (iii)because the organisms must be able to growunder anaerobic conditions more strict thanthose we are able to provide on a routine basis invitro, if they are to be competitive in the colon;and (iv) because we have observed that somespecies of facultative bacteria such as Esche-richia coli, lactobacilli, and some streptococcifrom other sources, including infections, canoften be recovered in higher numbers understrictly anaerobic conditions than they canunder aerobic conditions. In these latter cases,the microenvironment of the infection is usuallyanaerobic, as is the environment of feces.

It is most significant that many of the morenumerous fecal organisms have not been re-ported from human infections. B. fragilis ss.fragilis is by far the most common anaerobe inclinical infections of soft tissue, but here itoccurred at only 0.6% of the fecal flora. It isevident that the body defenses control andeliminate several hundred kinds of bacteria thatmay enter tissue after bowel trauma, becauseonly a relatively few are capable of initiatinginfection. These present observations indicatethat previous reports (49) of anaerobes occur-ring as predominant organisms in soft tissue in-fections as frequently as facultative organismsdo not result from contamination detected withimproved anaerobic methods. If that were thecase, the more numerous (but less pathogenic)species would have been found. Direct micro-

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TABLE 3. Relative frequency of bacterial species of the normal fecal flora of 20 Japanese-Hawaiians

Rank Counta [ Florab Organism(s)

(x 1010)1 5.76 (.49) 12.1 (1.0) B. fragilis ss.vulgatus2 3.40 (.38) 7.15 (.79) F. prausnitzii3 3.07 (.36) 6.45 (.75) B. adolescentis4 2.86 (.34) 6.02 (.72) E. aerofaciens5 2.65 (.33) 5.58 (.70) P. productus-II6 2.11 (.30) 4.45 (.62) B. fragilis ss. thetaiotaomicron7 1.74 (.27) 3.67 (.56) E. eligens (39)8 1.58 (.26) 3.32 (.54) P. productus-I9 1.53 (.25) 3.23 (.53) E. biforme10 1.16 (.22) 2.45 (.46) E. aerofaciens-HI

11-12C 1.12 (.22) 2.36 (.45) E. rectale-I, B. fragilis ss. distasonis13 1.08 (.21) 2.27 (.44) E. rectale-ll

(x 10')14 9.97 (2.0) 2.10 (.43) B. fragilis ss. a15 9.14 (1.9) 1.92 (.41) E. rectale-IV16 8.73 (1.9) 1.84 (.40) B. longum17 8.32 (1.8) 1.75 (.39) "Budding coccus" of Gossling18 7.08 (1.7) 1.49 (.36) B. infantis19 6.67 (1.7) 1.40 (.35) R. bromii20 6.26 (1.6) 1.32 (.34) L. acidophilus21 5.44 (1.5) 1.14 (.32) B. breve22 5.02 (1.4) 1.06 (.30) R. albus

23-24 3.80 (1.2) .799 (.26) B. fragilis ss. b, E. rectale-m-F25-28 3.39 (1.2) .713 (.25) E. ventriosum, B. fragilis ss. ovatus, R. torques (39), S.

hansenii (39)29 2.98 (1.1) .628 (.23) B. fragilisss. fragilis

30-34 2.17 (.94) .458 (.20) E. aerofaciens-II, Eubacterium U, L. leichmannii, C. catus(39), C. comes (39)

35-42 1.77 (.84) .374 (.18) E. rectale-HI-H, B. bifidum, E. coli, S. salivarius, B.fragilis ss. d, R. callidus (39), Ruminococcus AB, C.eutactus (39)

43-49 1.38 (.74) .291 (.16) E. formicigenerans (39), Eubacterium AK, L. salivariusvar. salicinius, B. clostridiiformis, B. fragilis ss. c,Bacteroides L, F. russii

(x 10')50-58 9.94 (6.2) .209 (.13) C. ramsoum-I, P. acnes, B. fragilis ss. e f, g, B.

hypermegas-I, S. intermedius (39), Peptococcus M,Ruminococcus P

59-75 6.21 (4.8) .131 (.10) E. ruminantium, Eubacterium-N-1, AG, L. rogosae (39),C. innocuum, Clostridium D, S. faecalis, S. faecalis var.liquefaciens, S. epidermidis, B. capillosus, B. fragilis ss.h, B. hypermegas, B. ruminicola s8. brevis, BacteroidesG, F. symbiosum, A. fermentans, Ruminococcus AJ

76-113 2.76 (3.1) .058 (.06) E. limosum, E. hallii (39), Eubacterium N, T, Z, AB, AE,AN, AQ, AY, B. pseudolongum, Bifidobacterium C, L.casei var. alactosus, L. fermentum, L. minutus, C.sporosphaeroides, C. nexile (39), C. ramosum, Clostrid-ium A, Escherichia "group," E. hafnia, K. pneumoniae,B. furcosus, B. eggerthii (39), Bacteroides J, K, L-1, L-3,AA, F. mortiferum, Fusobacterium 0, P. asac-charolyticus, Ruminoccocus AL, AQ, AU, BP,Coprococcus (39) AN-H, BN-1

aThe estimated count per gram of fecal dry matter (±- standard deviation of the estimate) is given.'The percentage of fecal population (± standard deviation of the percent) is given.c Whtre two rank numbers are listed, each organism cited was detected with equal frequency.

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scopic observations of clinical material, therelatively few (1 to 7 or 8) types of bacteriaisolated from such individual infections, andthe more limited range of species found inclinical infections further suggest that only aminority of the organisms in the intestinal florais pathogenic.The extremely dense and heterogeneous pop-

ulation of bacteria in feces was the subject ofmuch interest and conjecture, even before it wasknown that nearly all, if not all, of the bacteriaobserved are viable, metabolizing cells. Sugges-tions have been made that a major source ofnutrient for these organisms includes sloughedepithelial cells and other host protein materials.However, proteolytic activity was noticeablyabsent or very weak among the predominantorganisms encountered in the present study.Ammonium salts provide a suitable nitrogensource for several of the most numerous species,and specific carbohydrates apparently are es-sential for growth of many others.On the basis of these observations, we might

expect the type and amount of complex carbo-hydrate in the diet, or mucin, to exert animportant controlling influence upon the com-position of the flora. On the basis of work in ourlaboratory by Diwan Singh (M.S. thesis, Vir-ginia Polytechnic Institute, Blacksburg, 1968),we may further expect the effect of changes inthe diet to be quite complex. He found thatseveral intestinal isolates of predominant spe-cies inhibit the growth of other intestinal spe-cies. Thus, changes in diet may either stimulatecertain groups or fail to support growth ofspecies that normally control others. Perhaps,as careful attention is given to individual prop-erties and relative numbers of the bacteriapresent, some basic principles regarding therelationship between the flora and its host andenvironment can be established.

Identification of intestinal isolates. Wehave accepted more variation in characteristicsof certain "species" than of others, because,whereas some groups of strains are extremelyuniform in characteristics, other groups form acontinuum of phenotypes with no obvious divid-ing lines. Every attempt has been made torecognize the distinctly different organisms en-countered, and the descriptions reported hereare based on the best information available tous now. However, they may require furthermodification as additional detailed studies ofthe isolates are performed and more examples ofeach are encountered.New species. The new named species in

Tables 2 and 3 include only those for which wehad nine or more isolates from at least six

persons in this and related studies. Descrip-tions of these species and of the genus Copro-coccus are being published elsewhere (39).Previously described species. Although we

used procedures described in the AnaerobeLaboratory Manual (38) to identify the isolates,the species described there are mostly thosethat also occur in clinical specimens. Therefore,the number of described species and subspeciesis relatively limited, and the number of testsrequired for their differentiation is likewiselimited. There are many more species found inthe intestinal flora, some of which are quitesimilar to those previously described, but whichcan be shown to differ in a number of propertiesif they are examined in more detail. On thebasis of many new intestinal isolates in this andrelated studies, some additional description anddifferentiation of previously described species isnecessary.Eubacterium. Some strains of Eubacterium

aerofaciens are very similar to strains of Strep-tococcus intermedius. We have differentiatedthese species primarily on the basis of morphol-ogy and hydrogen production. All cells of S.intermedius are nearly spherical, whereas somecells in cultures of E. aerofaciens are ovoid ordefinite rods. Rods with swellings in the center("pelton de jardinier" or gardener's ball ofstring on a stick), as mentioned in the originaldescription by Eggerth (18), are sometimesproduced in peptone yeast extract (PY) basalmedium and help in recognition of the species.All strains of S. intermedius produce acid fromcellobiose, but this characteristic is variable inE. aerofaciens. A cotype strain of S. intermediusproduces no hydrogen. Strains of E. aerofaciensproduce copious hydrogen. Many strains of E.aerofaciens produce large amounts of formicacid. We recognize three phenotypic groups ofE. aerofaciens. Those labeled "E. aerofaciens-II" uniformly do not ferment sucrose; thoselabeled "E. aerofaciens-HI" uniformly do notferment cellobiose and produce little or no acidin salicin.Eubacterium biforme strains conform well to

the original description by Eggerth (18). Thisspecies is well named. Definite rod-shaped cellsmay not appear in all cultures of each strain,but, when they do, observation of cocciattached to rods is the only assurance that theculture is not a mixture. The production ofcaproic acid in small amounts is a most distin-guishing characteristic, but sometimes is onlydetected on repeated culture.Eubacterium cylindroides: See discussion

under Fusobacterium prausnitzii (below).Eubacterium rectale strains fall into at least

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five distinct groups. All strains ferment cellobi-ose, fructose, glucose, lactose, maltose, andstarch. They decolorize readily, and gram-posi-tive cells cannot be demonstrated in somestrains. The rods are usually curved and usuallymotile or have flagella singly, in pairs, or intufts near one or both ends of the cells. Butyricacid is a major product, together with variableamounts of lactic acid. Strains labeled "E.rectale-I" are slender curved rods, generallylonger than the other phenotypes, frequentlywith large central or terminal swellings. Theyuniformly ferment arabinose, melezitose, meli-biose, raffinose, sucrose, and xylose, and pro-duce large quantities of hydrogen. Strains of F.mortiferum may be similar to "E. rectale-I",except that they are thicker rods showing morepleomorphism, especially on blood agar, andhave no flagella.

Strains labeled "E. rectale-II" differ from "E.rectale-I" strains in that they fail to fermentmelezitose and may or may not ferment melibi-ose and sucrose. They are generally shorter,more uniform, curved rods.

Strains labeled "E. rectale-El-H" differ from"E. rectale-ll" strains in that they fail toferment raffinose. Heat-resistant spores havebeen detected in some strains otherwise similarto these three types of E. rectale. The strainswith spores have been designated as"Clostridium-A" (see Table 5). Many strainswith swellings, however, do not resist heating at80 C for 10 min, and are believed to beEubacterium species. Strains labeled "E. rec-tale-Ill-F" have the same reactions as "Ill-H"strains, except that they produce majoramounts of formic acid and no hydrogen. Wehave not demonstrated spores in any strains ofthis type. Strains labeled "E. rectale-IV" aresimilar to "E. rectale-I-H" strains. However,they fail to ferment xylose, may or may notferment raffinose, and never reduce the pH inarabinose to below 5.5. The relationship of theE. rectale strains to Butyrivibrio fibrisolvens isin question. A number of the different variantsof B. fibrisolvens from the rumen of cattle thatBryant and Small (10) described correspondclosely to some E. rectale strains described herewhich do not stain gram-positive. However, thereference strains of B. fibrisolvens generally areeven more fastidious than these isolates fromhumans. The isolates from humans are lessaerotolerant than most other human intestinalspecies, as reported by Attebery et al. (5). Thereference strains of B. fibrisolvens that we haveexamined are monotrichous and never staingram-positive, even in 4- to 6-h cultures. Bryant(personal communication) states that, rarely, a

rumen strain presumed to be a Butyrivibriospecies may stain weakly gram-positive.

Strains labeled E. ruminantium and E.ventriosum are very similar. On the basis ofreactions of reference strains, we have desig-nated those that ferment cellobiose and produceno hydrogen as E. ruminantium. These strainsuniformly fail to produce acid in mannose, butusually produce acid from salicin. Strains of E.ventriosum either do not ferment cellobiose orthey produce hydrogen. They often fermentmannose and do not ferment salicin. Both ofthese species are coccoid to short rods. Thereactions and products (major formic acid, withvariable amounts of butyric, lactic, acetic, andsuccinic acids) are similar to those we obtainedwith the "budding coccus" of Gossling (J.Gossling, Abstr. Annu. Meet. Amer. Soc. Mi-crobiol., 1972, M 7, p. 81); however, the cells ofthe budding coccus are more nearly spherical,as are the attached "buds." Cultures of all threespecies may stain weakly gram-positive. Gossl-ing demonstrated that the cell wall structure ofthe "budding coccus" is gram-negative; the twoEubacterium species are considered to be gram-positive.

Lactobacillus. Many intestinal isolates ofLactobacillus acidophilus, L. Ieichmannii, andother named species of Lactobacillus fail togrow on aerobic plates, even after several trans-fers in anaerobic media. None of the referencestrains of named species or strains of lactoba-cilli from the intestinal tract produced hydro-gen. Strains of some other species (E.aerofaciens and Clostridium ramosum) maydiffer only slightly in morphology or culturalreactions, but they produce larger (moderate)amounts of acetic and formic acids and majoramounts of hydrogen.

L. leichmannii: Also, see discussion under C.ramosum (below).Bacteroides. Bacteroides fragilis includes a

number of intermediate phenotypic groups thathave not been described as subspecies previ-ously. The differential characteristics of thesegroups are given in Table 4.

Bacteroides hypermegas was found in onefecal sample. In another sample, we isolatedstrains with the same biochemical reactions andproducts, including very large amounts of propi-onic acid, but the cells are not of the large sizecharacteristic of B. hypermegas. We have la-beled these smaller strains "B. hypermegas-I"and suspect that they are a distinct species.

Bacteroides ruminicola ss. ruminicola and ss.brevis are limited to those strains that do notproduce hydrogen. A number of strains withbiochemical characteristics similar to those

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TABLE 4. Characteristics of other subspecies groups of B. fragilis*

Characteristic

No. strains

AmygdalinArabinoseCellobioseEsculinGlycogen

MannitolMannoseMelezitoseMelibioseRaffinose

RhamnoseRiboseSalicinSorbitol

Starch pHStarch hydrolysisTrehaloseXylose

Gelatin digestionIndoleHydrogen

Subspecies group

a 'b' 'c' 'd' 'e' 'f' 'g' 'h

75 27 4 7 3 5 9 2

v a - w- - -w a- w-a a - aw wa - a- a-w a - w- a - aw wa- a- w w wa - w- wa- a a nt nt - a- a

- a - - - - _ -

aw a a aw wa aw v a- -a - -a - w-w a- aw w nt aw awwa a a a a v a -w

wa v - aw a w -w av -a - nt nt - - a-w a - nt nt - -a- a- - nt nt

a a a - a wa aw a+- + + - +- - +- +

- - - a- a a - awaw a a a a aw a- a

-w

-,2

+- -w -+ -w +- v +w-_ -_ + - 2 +

- nt 1 nt nt 2,3 nt

All strains are non-motile and produce acid from fructose, glucose, lactose, maltose, and sucrose. Theycoagulate milk, hydrolyze esculin, grow well in PYG-bile broth, and do not reduce nitrate, producecatalase, or digest milk. No strain tested (representatives of subspecies 'a', 'b', 'c', 'f, 'g', 'h') producesacid from erythritol or inositol.

* Abbreviations and symbols: a (carbohydrate cultures), acid (below pH 5.5); +, positive reaction; -,negative reaction; nt, not tested; v, variable reaction; w, weak reaction or pH between 5.5 and 6.0; numbers(growth and gas), amount estimated on "- to 4+" scale. Where two reactions are given (e.g., aw), the first is themore usual and the second is observed less frequently.

listed for these two subspecies were found, andgrowth was inhibited or not stimulated by bileafter transfers directly from the isolation me-dium. However, many of these strains producedhydrogen, and upon retesting their growth wasstimulated by bile; they were therefore identi-fied as B. fragilis.

Butyrivibrio. Butyrivibrio fibrisolvens: Seediscussion under E. rectale (above).Fusobacterium. Based on the study of many

new isolates, emended descriptions of F.prausnitzii and E. cylindroides, organismswhich are quite similar in many respects, arebeing published elsewhere (11, 12). Strains of F.prausnitzii generally fail to ferment carbohy-drates, although one or several may be weaklyfermented. All strains hydrolyze esculin astested by ferric ammonium citrate, and theyproduce little or no gas. Cells are usually stoutwith rounded ends, may have swellings, andoften stain unevenly. They are not motile andproduce no hydrogen. The major fermentationproducts include butyric acid with variable

amounts of formic and lactic acids and oftenminor or trace amounts of acetic, succinicand/or pyruvic acids and ethanol.

Strains of F. prausnitzii are differentiatedfrom E. cylindroides, which decolorizes easily,in that all strains of E. cylindroides produceacid (below pH 5.5) in mannose, usually inglucose and/or fructose, and sometimes in sali-cin and sucrose (11). F. prausnitzii is mosteasily differentiated from F. mortiferum in thatF. mortiferum produces major amounts of gasand acid in a number of carbohydrate media. F.prausnitzii is most easily differentiated from F.russii in that F. russii does not hydrolyzeesculin.

Clostridium. Clostridium ramosum and "C.ramosum-I" have similar reactions and mor-phology; however, strains labeled C. ramosumproduce large amounts of hydrogen and usuallyferment melibiose and raffinose, whereas "C.ramosum-I" does not. "C. ramosum-I" differsfrom L. leichmannii in producing large amountsof formic acid as the major acid product.

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Although cell size is similar in many cultures,thick cells are not observed in any cultures of"C. ramosum-I" as they may be in youngcultures of L. Ieichmannii. Both species produceD(-) lactic acid.Cocci. Acidaminococcus fermentans and

Peptococcus prevotii have a similar appearanceand similar reactions and, as has been noted byothers, both may stain weakly gram-positive.Strains of P. prevotii (including the type strain)become weakly fermentative if Tween 80 isadded to glucose or fructose, whereas strains ofA. fermentans remain nonfermentative.Budding coccus of Gossling: See discussion

under E. ruminantium (above).Peptostreptococcus productus strains form a

continuum of phenotypes. All strains hydrolyzeesculin and ferment glucose, fructose, raffinose,and xylose and a majority of the other carbohy-drates tested. Individual strains may fail toferment a few of the other carbohydrates. Hy-drogen production is variable (usually largequantities are produced). All strains produceacetic acid, usually as the major product. Moststrains also produce variable amounts of suc-cinic and lactic acids. Ethanol and pyruvic acidmay be produced in small amounts. The cells insome cultures become extremely elongate andmay appear as tapered rods. Typically, pointedoval (football-shaped) cells occur in chains. Thestrains labeled "P. productus-I" produce mod-erate to large amounts of formic acid anduniformly ferment lactose. The strains labeled"P. productus-ll" do not uniformly fermentlactose, but, unlike P. productus-I, they uni-formly ferment arabinose, maltose, and sucrose,and they do not produce formic acid.

S. intermedius (39): See discussion under E.aerofaciens (above).The identification of facultative cocci listed

here was based on direct comparison of resultsfrom labeled ATCC strains characterized inanaerobic media with results from the faculta-tive cocci obtained in this study, using the sameculture procedures as for anaerobes. Identity ofthe ATCC strains was verified by comparingresults with published descriptions.Groups or strains that do not belong to

described species. Cultural reactions of strainsnot belonging to recognized species or groupsdiscussed above are given in Table 5 and thediscussion following. In addition to the charac-teristics given in Table 5, the strains did not:produce acid from dulcitol, grow in PY-6.5%NaCl-glucose broth, produce propionate fromthreonine, or produce acetylmethylcarbinol(AMC), unless specifically stated below. Also,unless otherwise indicated, neutral red wasreduced, and growth was as good in routine

prereduced broth medium supplemented withheme and vitamin K as it was in medium withrumen fluid (10 or 30%) or Tween 80 (0.01%)added. No strain grew on the surface of bloodagar plates incubated in a candle jar or aerobicatmosphere.

In the characteristics given below, the deepagar colonies are in prereduced RGCA roll tubesafter incubation for 5 days. Surface colonieswere observed on brain heart infusion agar orSweet E agar streak tubes or on anaerobic sheepblood agar plates after incubation for 1 to 2days.Eubacterium. Eubacterium-N: Deep agar

colonies are pinpoint to 0.5 mm in diameter,tan, lenticular, and translucent. Agar surfacecolonies are 0.5 to 1 mm in diameter, circular,convex or flat, entire, opaque, dull, and smooth.

Glucose broth cultures have sediment thatadheres to the bottom of the tube and usuallyhave no turbidity; the pH is 5.8 in 5 days.Eubacterium-N-1: Deep agar colonies are 0.2

to 1 mm in diameter, white, lenticular, andtranslucent. Agar surface colonies are 0.5 to 1mm in diameter, circular, entire, convex,opaque, dull, and smooth.

Glucose broth cultures have ropy sedimentwithout turbidity; the pH is 5.7 to 6 in 5 days.Tween 80 (0.01%) is required for maximumgrowth; growth is produced in PY-6.5% NaCl-glucose broth.Eubacterium-T: Deep agar colonies are 2 mm

in diameter and rhizoid. Agar surface coloniesare 4 mm in diameter, circular, entire, opaque,white, shiny, and smooth.

Glucose broth cultures have a stringy, mucoidsediment, usually with no turbidity; the pH is 5in 5 days. AMC is produced.Eubacterium-U: Deep agar colonies are 1 to 4

mm in diameter and of "raspberry" form.Surface agar colonies are 1 to 4 mm in diameter,circular, flat to low convex, translucent, anddull.

Glucose broth cultures have stringy (some-times grainy or smooth) sediment and usuallyhave no turbidity; the pH is 5 to 5.4 in 5 days.Neutral red is not reduced by three of the fivestrains tested.

Eubacterium-Z: Deep agar colonies are 1 to 2mm in diameter and appear as fluffy "woolyballs." Agar surface colonies are 1 to 2 mm indiameter, circular, low convex, entire, and gran-ular.

Glucose broth cultures are turbid withsmooth or stringy sediment; the pH is 4.9 to 5.3in 5 days. AMC is produced.Eubacterium-AB: Deep agar colonies are 0.5

mm in diameter, white, and lenticular. Agarsurface colonies are 3 to 5 mm in diameter,

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972 MOORE AND HOLDEMAN APPL. MicROImOL.

TABLE 5. Characteristics of less common bacteria of the fecalflora*

-~~~~~~m * O z aZ Z 1 D N < < < < ( '

Characteristic .T. .3.A. > M A

M M < .2 UL CL q< <N_

XJ

LzX Lr~ LQ W W LQ LW W W L

No. strains 4 6 1 5 6 2 3 3 6 4 1

Amygdalin - w- w - -w -w - - - w -

Arabinose - - - -w - a aw - - w -

Cellobiose - - - aw - a aw - - a -

Dextrin - - - - - -w - - - w- -Erythritol - - - - - - - - - -

Esculin pH - - - - -w wEsculin hyd. - - - + +- + + - - +Fructose wa -w - a a a W aw - a -wGalactose -w - w a wa aw - - - aGlucose w- w- a a a a -w - - a a

GlycerolGlycogenInositol - - - - a - - --Inulin - - - - aw a - - - aLactose - - - a a- a -w - v

Maltose - - a aw v a - - - aMannitol - - - -w -wMannose v - a w- a - - - - wMelezitose - - - - - - - - - - -

Melibiose - - - aw -w w

Raffinose - - - a - a - - - -aRhamnose -a - - - - - - - - - aRibose - - - - - - - - - - aSalicin -w - - aw v aSorbitol - - - v -w a

Sorbose - - w - awStarch pH - - - - - - - -w - w-Starch hyd.Sucrose - -w a v a a - - - aTrehalose - - - - vXylose - - - - aw - aw - - a-

Gelatin dig. - - - - - - - - w-Milk - - - - -c - c - - -c -Indole - - - - - - - - - - -

EYA react. - - -

HemolysisH2S - - - _ - w - - -_ - -

Gas 1 - 1 4 3,4 4 v - - 4 4Hydrogen 4 - 1 4 4 4 4 - - 4 4PY-growth -,1 1 2 2,1 2 2,3 -,1 1 1,- - 2PY-CHO gr. 2 4 4 4 4 4 2,3 2,3 1,- 4 4PYG-bile gr. 1 3 - 4 3,4 - 2,- 2,1 -,1 4 2,3Pyruvate (A) - AF AB B(A) AF - A(F) - - AGluconate - - - (AB)Motility - - - - - - v -,+ - - -

MorpologJ* (It Ct11;, ,fv10 ' ' "

I micronslI1tsC

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VOL. 27, 1974 HUMAN FECAL FLORA 973

TABLE 5. cont'd.

I _,--mnJ a0 0.>- z z 'U . < < co< 6 m sXco2...I

,~ ~~_., 'J;s_ z :

Characteristic . ' C a >

~~~. a~~~~U .- U-

u -, Qjx x x t

No. strains 2 1 1 4 4 5 3 1 2 1 4Amygdalin - - a - - v - a - - -

Arabinose a a -w - - - awCellobiose - - - - - - - aDextrin w w- w - - w- - - -wErythritol -wEsculin pH - w - - - w a -wEsculin hyd. - + + - + + - + + - +Fructose aw a a -w aw aw a a a aw aGalactose wa a a - a aw - a a - awGlucose aw a a - a a w a a w- awGlycerol vGlycogenInositol - - - - - - - -

Inulin - - - - -w wa - - -w - aLactose a- a - - - - - a a - aMaltose a a a - a a v a a - aMannitol aMannose w a a - - - -w - - - aMelezitose - -

Melibiose - - - - - w - - aw - aRaffinose - - - - - a - - v - aRhamnose - - - - - a a- - - - aRibose - - - - -w a -a - - - waSalicin - a a - - a - a -wSorbitol a - - - -wSorboseStarch pH - w w - -w w- - - -wStarch hyd. - - - - - +Sucrose - v a - -w a - - - - -wTrehalose - - - - - - - - - - -

Xylose wa - - -a aw - - - - aGelatin dig. - - - - - - - wMilk - c- - - - - -- c - - -cIndoleEYA react.Hemolysis b- - - -a-H2S - - - + - v - - - - -

Gas -,1 2 3 3 3 3,4 - 1 3 2,3 3Hydrogen 4 3 2 4 4,2 4 - - 4 4 4PY-growth 2 1,- 1 2 1,- 1,2 1,- -,1 2 1 1,-PY-CHOgr. 4 4 3 4 4 4 4 4 4 4 4PYG-bile gr. 3 3 3 v 4,- 4 - 3 4 3 4Pyruvate A(S) - - CA+ AF AF - AF A A(-)Gluconate - - - - - AFMotility - - - - - - - - - -

Morphology ' S /

10 '' S j,micronsI) ;~: *

%6 too~~~~'A:~~~~~'~~~~~~~~~~ ID g~ ~~~~~Sl

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TABLE 5. cont'd.

0 0

O_~~~~n i J J ~J <. ,, . .

Characteristic > , ,_,,_

No. strains 2 1 1 6 2 4 3 1 2 5 1

Amygdalin - - - - - - wArabinose - - -w - - - -w - w - -Cellobiose - a - - - - - - a - aDextrin - w - - - - - - wErythritol - - - - - - - -

EsculinpH - - - - - - - - -wEsculin hyd. v + + - - - + + +Fructose w- - aw -w - - a w a aw aGalactose - - a w- - - a w aw aGlucose w- - aw w- - - a w a a

Glycerol - - - - - - - wGlycogenInositolInulin - - - _- - - - _Lactose -w a - - - - -w - a a-

Maltose - a a - - - a - a waMannitol - - - - - - - -w - vMannose w- - a - - - a w a aMelezitose - - aw - - - - - vMelibiose - - w - - - - - wa

Raffinose -w - a - - - a- - awRhamnose - - aRibose wSalicin w a - - - - - - -wSorbitol - - - - - - - - - -w

SorboseStarch pH -w a - - - - - - w - aStarch hyd. - - - _ _ - + - +Sucrose - - a -w - - a - a aTrehalose - - w - - - wa - -wXylose -w - a - - - a- - a

Gelatin dig. - - - +_Milk - c - - - - - - c c- -Indole - - - - - - + +EYA react.Hemolysis - - - - - - - a b- a-

H2S - - + - - + +- + - - -

Gas -,2 - 4 - - - 1 4 4 4Hydrogen - - 4 -,1 - 2,- - 4 4 4PY-growth 1,2 -,2 3 2,- -,1 1,3 3 3 1 1 1PY-CHO gr. 3 4 4 2,- -,1 1,3 4 4 4 4 4PYG-bile gr. 2,3 - 4 - - 1,2 4 4 1,2 4 1,3Pyruvate - - A A(L) A(F) AB BA A AGluconate - - AF -(A) - - A(F) - - - AFMotility - - + - - - - - + - -

ofi0 J) @ /| , \~~~~~~I -Morpholog A

lo d'I

Imicrons I j'~ '/( ~ l/~)U'-9

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circular, slightly erose, convex, opaque, slightlymottled, smooth, shiny, and mucoid.

Glucose broth cultures have fluffy or powderysediment and usually have no turbidity; the pHis 4.7 to 5 in 5 days. AMC is produced by one ofthe two strains tested.Eubacterium-AE: Deep agar colonies are 0.5

mm in diameter, lenticular, and white. Agarsurface colonies are pinpoint, circular, entire,low convex, slightly mottled, smooth, and shiny.

Glucose broth cultures are turbid and have asmooth sediment; the pH is 5.7 to 6 in 5 days.Arabinose and xylose are more strongly fer-mented, with a pH value of 5.2 to 5.8 in 5 days.Growth is stimulated by glucose, fructose, lac-tose, and cellobiose, although the pH may notdecrease very much in these media.One of the three strains is motile with one to

three subpolar flagella.Eubacterium-AG: Deep agar colonies are

minute to 0.5 mm in diameter, lenticular, andwhite or tan. Agar surface colonies are pinpointto 1.5 mm in diameter, circular, entire to lobate,low convex, translucent, white, shiny, andsmooth.

Fructose broth cultures produce smooth sedi-ment with no turbidity, or very little turbidity;the pH is 5 to 5.7 in 5 days.One of the three strains is motile and peritri-

chous.The utilization of pyruvate, lack of hydrogen

production, and larger cell size differentiatethese organisms from similar organisms (iso-lated in another study; manuscript in prepara-tion), which we have designated Eubacterium-AG-H.Eubacterium-AK: Deep agar colonies are 0.1

mm in diameter, white or translucent, andlenticular to bifoleate. Agar surface colonies arepinpoint to 0.5 mm in diameter, circular, andtransparent.

Glucose broth cultures produce a slightlystringy sediment and 6ften have no obvious

turbidity. The cells may decolorize easily, andonly some elements in a chain or some portionsof a cell may stain gram-positive. Rumen fluid(30%) is required for growth.Eubacterium-AN: Deep agar colonies are 0.5

to 1 mm in diameter, white, and fuzzy or"cauliflower-like." Agar surface colonies aregranular, erose, flat to slightly umbonate, trans-lucent, tan, and spreading.

Glucose broth cultures have a smooth sedi-ment and only slight turbidity; the pH is 4.8 to 5in 1 day. AMC is produced.Eubacterium-AQ: Deep agar colonies are 0.5

mm in diameter, lenticular, and tan. Agarsurface colonies are pinpoint (on anaerobicsheep blood agar plates) to 2 mm in diameter(on streaked roll tube), circular, entire, semi-opaque, shiny, and smooth.

Glucose broth cultures have smooth to stringysediment, and often have no turbidity; the pH is4.8 to 5.5 in 1 to 2 days. Although the cellularmorphology is quite distinctive, not all fecalstrains with the "O," "S." and coiled shapeshave the other characteristics of this strain.Similar morphology may be observed, for exam-ple, in some strains of C. ramosum.Eubacterium-AY: Deep agar colonies are 0.5

mm in diameter, opaque, and lenticular to"fuzzy." Agar surface colonies are pinpoint,circular, entire, semi-opaque, and yellowish.

Fructose broth cultures have smooth- sedi-ment and produce a slight turbidity; the pH is5.3 to 5.6 in 5 days. Dulcitol is strongly fer-mented; the pH is 5.2 to 5.4 in 5 days.Coprococcus. Coprococcus-AN-H: Deep agar

colonies are 0.5 mm in diameter, lenticular, andtan. Agar surface colonies are 1 to 2 mm indiameter, circular, entire, semi-opaque, shiny,and smooth.

Glucose broth cultures have powdery sedi-ment and no turbidity; the pH is 5 in 1 day. Theproduction of AMC and growth in PY-6.5%NaCl-glucose broth are variable.

* Acids and alcohols produced in PY-carbohydrate (usually glucose or fructose) broth cultures are given inbrackets after the name of the organism. Those produced from pyruvate and lactate are indicated in the table.Capital letters indicate 1 meq (or more) per 100 ml; small letters indicate less than 1 meq/100 ml. Products inparentheses are not uniformly detected. Abbreviations for products: a, acetic acid; b, butyric acid; c, caproicacid; c', caprylic acid; f, formic acid; h, heptanoic acid; ib, isobutyric acid; iv, isovaleric acid; 1, lactic acid; p,propionic acid; py, pyruvic acid; s, succinic acid; v, valeric acid; 2, ethanol; 4, butanol.

Abbreviations and symbols for other reactions: a (carbohydrate cultures), acid (below pH 5.5); a (hemolysis),alpha; b (hemolysis), beta; c (milk), curd; +, positive reaction; -, negative reaction; -, no visible growth oronly slight growth and negative reaction; nt, not tested; v, variable reaction; w, weak reaction or pH between 5.5and 6.0; numbers (growth and gas), amount estimated on "- to 4+" scale. Where two reactions are given (e.g.,'aw'), the first is the more usual and the second is observed less frequently. EYA, Egg yolk agar (modifiedMcClung-Toabe).No strain tested produces acid from adonitol, utilizes lactate, produces catalase, reduces nitrate, or digests

meat.

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Coprococcus-BN-1: Agar surface colonies are0.5 to 2 mm, circular, entire, flat to umbonate,colorless, shiny, and smooth.

Glucose broth cultures are turbid and have asmooth sediment; the pH is 5 in 1 day.Peptococcus. Peptococcus-M: Deep agar col-

onies are 0.5 to 1 mm in diameter, white, andlenticular. Agar surface colonies are 0.5 to 1 mmin diameter, circular to irregular, convex, entireto erose, and dull.

Glucose broth cultures are turbid and have aropy sediment.Ruminococcus. Ruminococcus-P: Deep agar

colonies are 0.5 to 1.5 mm in diameter, lenticu-lar, translucent, and tan. Agar surface coloniesare 1 to 5 mm in diameter, translucent toopaque, buff to white, entire to erose, convex toumbonate, and smooth.

Glucose cultures are turbid and have asmooth to stringy sediment; the pH is 5 to 5.5in 1 to 2 days. Growth in PY-6.5% NaCl-glu-cose broth is variable.Ruminococcus-AB: Deep agar colonies are 0.5

to 2 mm in diameter, white, and lnticular.Agar surface colonies are 0.5 to '1 mm indiameter, circular, entire, low convex, andtranslucent.

Glucose broth cultures are turbid and have aheavy smooth sediment; the pH is 4.8 to 5 in 1to 2 days. Dulcitol is weakly fermented by twoof the five strains.

Ruminococcus-AJ: Deep agar colonies are 0.5to 1 mm in diameter, lenticular, tan, andtranslucent. Agar surface colonies are 0.5 to 1.5mm in diameter, circular to slightly irregular,convex or slightly raised, translucent, shiny,and smooth.

Fructose broth cultures are turbid and have asmooth sediment; the pH is 4.7 in 5 days.Ruminococcus-AL: Deep agar colonies are 1

mm in diameter and lenticular. Agar surfacecolonies are 1 to 2 mm in diameter, circular,entire, convex, granular, dull, and opaque.

Glucose broth cultures have flocculent sedi-ment and no turbidity; the pH is 4.8 in 1 day.Ruminococcus-AQ: Deep agar colonies are 2

to 5 mm in diameter, lenticular, with opaque,tan centers and irregular translucent edges.Agar surface colonies are 0.5 mm in diameter,circular, entire to erose, low convex, slightlygranular, semi-opaque, and buff.

Glucose broth cultures have a flocculent toropy sediment and sometimes have no turbid-ity; the pH is 5.2 to 5.5 in 1 day. Hippurate ishydrolyzed.Ruminococcus-AU: Deep agar colonies are 1

mm in diameter, white, and lenticular. Agar

surface colonies are 0.5 mm in diameter, circu-lar, entire, convex, grayish, and smooth.

Fructose broth cultures have a smooth sedi-ment and no turbidity; the pH is 5 afterincubation for 5 days.Ruminococcus-BP: Deep agar colonies are 1

mm in diameter, lenticular, and tan. Agarsurface colonies are 0.5 to 1.5 mm in diameter,circular, entire, low convex to flat, translucent,and smooth.Glucose broth cultures have smooth sediment

and no turbidity; the pH is 5 to 5.2 in 1 day.Bacteroides. Bacteroides-G: Deep agar colo-

nies are 0.5 mm in diameter, white, and lenticu-lar. Agar surface colonies on BHIA are 0.5 to 1mm in diameter, circular, entire, low convex,translucent, smooth, and shiny; on sheep bloodagar plates at 4 days, there is poor growth.Colonies are 0.5 mm in diameter, circular,erose, tan, fried egg-form, and have clear granu-lar edges and dense translucent centers. Thecultures produce no hemolysis.Glucose broth cultures have a crusty sedi-

ment and no turbidity; the pH is 5.8 to 6 in 5days.

Bacteroides-J: Deep agar colonies are 1 mmin diameter and lenticular. Agar surface colo-nies are 1 to 2 mm in diameter, circular, entire,convex, opaque, dull, granular, and smooth orbumpy.

Lactose broth cultures have a flocculent sedi-ment and very little turbidity; the pH is 5.2 to5.3 in 5 days. Hippurate is hydrolyzed.

Bacteroides-K: Deep agar colonies are 1 mmin diameter, white, and lenticular. Agar surfacecolonies are 0.5 to 1 mm in diameter, circular,entire, convex, translucent, shiny, and smooth.

Glucose broth cultures are turbid and have asmooth sediment; the pH is 5.1 in 1 day. Thecells are motile and have a single lateral orsubpolar flagellum.

Bacteroides-L: Deep agar colonies are minuteto 0.5 mm in diameter, transparent to translu-cent, lenticular, and white to tan. Agar surfacecolonies are pinpoint to 1.5 mm in diameter,circular to slightly irregular, flat to low convex,transparent to translucent, smooth, and shiny.

Glucose broth cultures have stringy to floccu-lent sediment and usually no turbidity. Rumenfluid (30%) somewhat enhances the growth ofmost strains. Gelatin liquefaction probably isdependent on the amount of growth. Severalstrains did not withstand lyophilization orfreezing at -70 C.The cellular morphology, generally better

(although poor) growth, slight fermentation of afew carbohydrates, and lack of isobutyrate or

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isovalerate production distinguish this groupfrom Bacteroides L-1. Lack of pyruvate utiliza-tion, poorer growth, and the slight carbohydratefermentation distinguish the group fromBacteroides-L-3.

Bacteroides-L-l: Deep agar colonies are 0.2mm in diameter and lenticular. We have beenunable to obtain surface growth of these strains.

Glucose broth cultures have very slight or noturbidity, but cells can be seen in Gram stainsand wet mounts.

Bacteroides-L-3: Deep agar colonies are 0.5 to1 mm in diameter, white to tan, and lenticular.Agar surface colonies are 0.5 to 1 mm indiameter, circular to slightly irregular, slightlyerose, raised, translucent, and yellowish.

Glucose broth cultures have a smooth sedi-ment and usually are turbid. The production ofH2S (SIM) and utilization of pyruvate distin-guish this group from Bacteroides L and Bacte-roides L-1.

Bacteroides-AA: Deep agar colonies are 0.5mm in diameter, lenticular, and translucent.Agar surface colonies are 0.5 mm in diameter,circular, entire, convex, and translucent.

Glucose broth cultures are turbid and have asmooth sediment; the pH is 5.4 in 5 days. AMCis produced.Fusobacterium. Fusobacterium-0: Deep

agar colonies are minute, white, and lenticular.Agar surface colonies are 2 to 4 mm in diameter,irregular, lobate, raised with concave center,and translucent.Glucose broth cultures have a smooth sedi-

ment and little turbidity; the pH is 5.7 to 5.9 in1 to 2 days. Threonine is converted to propio-nate.This organism differs from F. nucleatum by

morphological characteristics of the cells and byhydrolyzing esculin.

Clostridium. Clostridium-A: Deep agar colo-nies are 0.5 to 1 mm in diameter, tan, andtransparent. Agar surface colonies are 1 to 2 mmin diameter, circular, entire to erose, low con-vex, beige, and semi-opaque.

Glucose broth cultures are turbid with asmooth sediment; the pH is 5.2 to 5.5 in 5 days.

Oval, terminal (sometimes subterminal)spores are produced. The spores survive heatingat 70 C for 10 min. One of the two strainssurvived heating at 80 C for 10 min. Cells aremotile and have polar tufts of flagella.

Clostridium-D: Deel, agar colonies are 2 to 3mm in diameter, white, and lenticular. Agarsurface colonies are 2 mm in diameter, circular,entire, convex, semi-opaque, grayish-white, andsmooth.

Glucose broth cultures are turbid and have asmooth sediment; the pH is 5.2 in 5 days.Cultures survive heating at 80 C for 10 min, butno spores have been seen in stained smears.

Bifidobacterium. Bifidobacterium-C: Deepagar colonies are 0.5 mm in diameter, white,and lenticular. Agar surface colonies are 0.5 to 1mm in diameter, circular, erose, convex, granu-lar, and translucent.Fructose broth cultures have a powdery sedi-

ment and some turbidity; the pH is 5 in 5 days.ACKNOWLEDGMENTS

This work was financed primarily by contract no. 71-2427from the National Cancer Institute and grant no. 14604 fromthe National Institute of General Medical Sciences.We also wish to thank Ruth Beyer, Sarah Chalgren, Ruth

Chiles, Dale Charles, Linda S. Knight, Steven Lowe, WilliamMiles, Carolyn Salmon, Christina Spittle, John Strauss,Barbara Thompson, Grace Upton, Margaret Vaught, MichaelWeaver, Susan West, Tracy Wilkins, and others of ourlaboratory who performed work on or for this project and GaryGlober and Grant Stemmerman, Kuakini Hospital, Honolu-lu, and John Berg and Sidney Silverman, National CancerInstitute, for their continued help. We gratefully acknowledgethe assistance of I. J. Good, Statistics Department, VirginiaPolytechnic Institute and State University, Blacksburg, forconsultation and help with the design and statistical analysis.

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