cellulolytic and non-cellulolytic bacteria in rat - applied and

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1982, p. 1428-1434 Vol. 44, No. 60099-2240/82/121428-07$02.00/0Copyright C 1982, American Society for Microbiology

Cellulolytic and Non-Cellulolytic Bacteria in RatGastrointestinal Tracts

JOAN M. MACY,* J. ROBERT FARRAND, AND LARRY MONTGOMERYDepartment ofAnimal Science, University of California, Davis, California 95616

Received 1 June 1982/Accepted 10 August 1982

Lactobacillus and Bifidobacterium species were the predominant organismsisolated from small intestinal (jejunal) contents of rats, and lactic acid was the onlyorganic acid detected. The numbers of cellulolytic bacteria in small intestineswere low (approximately 103/g). The fermentation in ceca was different from thatin intestines, as, in addition to small amounts of lactic acid, high concentrations ofvolatile fatty acids were detected. The mixed cecal microflora was able to digestcellulose (pebble-milled Whatman no. 1) and cabbage. High numbers of cellulolyt-ic bacteria were found (0.5 x 108 to 12.2 x 108/g; 6% of total viable bacteria). Thepredominant celluloytic organism isolated was Bacteroides succinogenes. Rumin-ococcusflavifaciens was isolated from a few animals. The kinds and numbers ofthe predominant non-cellulolytic organisms isolated from rat ceca were similar tothose described by previous workers.

The presence of dietary fiber in the diet iscorrelated with lower incidences of coronaryheart disease, arteriosclerosis, colonic cancer,gallstones, diverticular disease, and diabetesmellitus (35, 36). Since dietary fiber consists ofcomplex carbohydrates of plant origin that mam-malian gut enzymes cannot digest, a number ofinvestigators have suggested that the beneficialeffects of fiber may be due not only to theirphysical or chemical nature but also to theirfermentation in the cecum and colon (35). Todetermine the extent of fiber degradation in thelower gut, we decided to attempt the enumera-tion and isolation of cellulolytic bacteria fromthe rat cecum. Emphasis was placed on cellulo-lytic bacteria because cellulose is the most diffi-cult of complex carbohydrates to degrade. Ifsuch organisms are found in significant num-bers, one can conclude that dietary fiber isprobably fermented in this environment.

Reports supporting the hypothesis that lowergut microflora ferment fiber resulted from workwith animals having ceca and with humans.Significant numbers of cellulose-degrading orga-nisms were found in the ceca of guinea pigs (11),rabbits (15), and horses (10). A cellulolytic orga-nism (a Bacteroides species; 108/g) was isolatedfrom one of five human fecal samples (1, 2).Additionally, van Soest et al. (39, 40) and Ehle etal. (12a) demonstrated that the mixed microbialpopulations from human feces can degrade di-etary fibers such as bran and cabbage. Thepresence of bran or cabbage in the diet didnot significantly affect the ability of fecal orga-nisms to degrade various kinds of fiber (12a).

Salyers and co-workers found that human co-lonic Bacteroides sp. can ferment a varietyof complex polysaccharides, including xylans,non-cellulosic glucans, pectins, galactoman-nans, arabinogalactans, mucopolysaccharides,and mucin glycoprotein (20, 30, 31). Finally,Chang and co-workers observed that the addi-tion of bran or corn bean to a diet increasedactivities of ,-glucosidase (2.5-fold) and ot-galac-tosidase (4-fold) in human feces (30; G. W.Chang, H. E. Fukumoto, C. P. Gyory, A. P.Block, M. J. Kretsch, and D. H. Calloway, Fed.Proc. 38:767, 1979; G. W. Chang, personal com-munication).

MATERIALS AND METHODSAnimals and diets. Male Sprague-Dawley rats were

obtained from the Department of Animal ScienceSmall Animal Colony, University of California, Davis.Immediately after weaning (3 weeks old), the rats werefed a stock diet (Ralston Purina Co., St. Louis, Mo.;crude fiber, "no more than 6%"). Food and waterwere available ad libitum. Rats used for experimentswere 6 to 8 weeks old (average age, 7 weeks).

Intestinal sampling and processing. Rats were anes-thetized with ether, and the intestines were exposedvia a midventral incision. The duodenal and ileal endsof the small intestine and the opening to the cecumwere ligated. The different areas of the intestine wereseparated from each other and placed in ice. The smallintestine and cecum were transferred to an anaerobicchamber (Coy Laboratory Products, Inc., Ann Arbor,Mich.). The contents of the middle portion (15 to 20cm) of the small intestine (jejunum) or the entirecontents of the cecum were emptied into small beakersheld on ice. After thorough mixing of the contents with

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MICROORGANISMS OF THE RAT GUT 1429

TABLE 1. Microbiological mediaa

Ingredient PY-CF PY-CF agar PY-CF-MS PY-CF-MS Dilution PMC PMCbroth broth agar broth broth agar

Solution Ab 167 ml 167 ml 167 ml 167 ml 167 ml 167 ml 167 mlSolution BC 167 ml 167 ml 167 ml 167 ml 167 ml 167 ml 167 mlPeptone 5.0 g 5.0 g 5.0 g 5.0 gYeast extract 10.0 g 10.0 g 10.0 g 10.0 gTrypticase (BBL) 5.0 g 5.0 g 5.0 g 5.0 gNaHCO3 5.0g 5.0g 5.0g 5.0g 5.0g 5.0g 5.0gCysteine-HCld 0.25 g 0.25 g 0.25 g 0.25 g 0.25 g 0.25 g 0.25 gNa2S *9H2Od 0.25 g 0.25 g 0.25 g 0.25 g 0.25 g 0.25 g 0.25 gVitamin K1 0.001 g 0.001 g 0.001 g 0.001 gHemin 0.0025 g 0.0025 g 0.0025 g 0.0025 gSugar mixturee +f +Agare 15.0g 15.0g 5.0 gIntestinal-cecal extracth 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 100 mlPMC' 6.7 g 5.0 gVitamin mixture1 +VFA mixturek +

a Final volume, 1 liter.b Solution A contained (in grams per liter): NaCl, 5.4; KH2PO4, 2.7; CaCl2 * 2H20, 0.159; MgCI2 * 6H20, 0.12;

MnCl2 * 4H20, 0.06; CoC12 * 6H20, 0.06; (NH4)2SO4, 5.4.Solution B contained (in grams per liter): K2HPO4, 2.7.

d Prepared as a cysteine * HCl-Na2S solution under an atmosphere of 100% N2 and autoclaved separately (22).A mixture of the following sugars (each 5%) was prepared anaerobically under 100% N2 and autoclaved

separately: glucose, maltose, cellobiose, xylose, fructose. Final concentration of each sugar was 0.2%.f + indicates that this mixture was added.g Oxoid purified agar.h The extract ("cecal" extract) of rat intestinal and cecal contents (from ceca and intestines stored at -20°C)

was prepared under an atmosphere of 100% CO2 by extracting 1 part contents with 2 parts water in a Waringblender. The suspension was centrifuged at 17,000 x g for 1 h at 4°C; the supernatant was transferred, under anatmosphere of 100%o C02, to roll tubes and autoclaved.'The PMC was prepared by pebble milling 20 g of Whatman no. 1 filter paper in 1 liter of distilled water for 16

to 17 h. This suspension was then autoclaved and stored at 4°C until use.i The vitamin mixture added was that of Scott and Dehority (32).k The VFA mixture was that used by Caldwell and Bryant in medium 10 (16), but at a fivefold lower

concentration.

a sterile wotden stick, approximately 1 g (wet weight)of material was transferred aseptically to a tube con-taining 9 ml of dilution medium and four glass beads.The tube was closed and removed from the anaerobicchamber. The intestinal material was mixed for 30 s ina Vortex mixer. The suspended material was seriallydiluted (10-fold) in dilution media, and 0.5 ml wasinoculated into the appropriate enumeration medium.All procedures were carried out under strict anaerobicconditions (18). Inoculated cultures were incubated at38°C. The procedure described above for cultivation ofcecal or intestinal material is similar to that used byHoldeman et al. for culturing human fecal bacteria(16). A 1-g amount of the remaining material from theintestine and cecum was extracted with 2 volumes ofdistilled water. After centrifugation, the supernatantwas frozen until it was analyzed for lactate and volatilefatty acids (VFA).Media for enumeration, isolation, and characteriza-

tion of bacteria. All media were prepared anaerobicallyunder an atmosphere of 100% CO2 according to themethod of Hungate (18). The medium used to enumer-ate and isolate viable bacteria from either the smallintestine or cecum of rats was the peptone-yeastextract-mixed sugars-"cecal fluid extract" (PY-CF-

MS) agar medium described in Table 1. This medium issimilar to the PY medium of Holdeman et al. (16),except that the mineral solution used was that of Vareland Bryant (41), and an extract of intestinal plus cecalcontents was present.The medium described above was chosen because it

simulates the conditions in the rat intestine and cecum.Preliminary experiments comparing this medium withthe same medium to which a vitamin and VFA solution(Table 1) had been added or with a rumen fluidmedium (18) (in which the peptone, yeast extract and"cecal fluid" of the PY-CF-MS medium were replacedby 30% rumen fluid) showed that there was no signifi-cant difference in numbers of organisms detected.

Dilution medium and the medium used for theenumeration of cellulolytic bacteria are shown in Ta-ble 1.

Cellulolytic organisms were isolated in a pebble-milled cellulose (PMC) agar medium (Table 1). Thismedium contained only 0.5% agar, thus allowing clearzones to form. To permit simple observation of clear-ings that formed, the cellulose concentration was0.5%, and only 3 ml of medium was added to each 1.6-by 15-cm roll tube.Organisms were characterized by using a PY-CF

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1430 MACY, FARRAND, AND MONTGOMERY

TABLE 2. Concentrations of VFA and lactic acid in intestinal and cecal contents of ratsa

Site Acetic Propionic Butyric Lactic

Intestine <0.5 <0.3 <0.2 18.3 t 5.9Cecum 71.7 t 12.2 32.2 t 4.8 16.8 t 3.6 5.9 ± 1.0

a Average from six animals sampled on different days. Values are expressed as micromolar concentration +standard error of the mean; n = 6. Percent water of cecal contents was 80.3 t 1.3.

broth medium to which the appropriate substrate was

added (Table 1).Enumeration and isolation of bacteria. After inocula-

tion, rolling, and incubation of the enumeration medi-um, numbers of each colony type were determined intubes containing 30 to 300 colonies. Representativecolonies of each type were then purified according tothe method of Hungate (18) by subculturing severaltimes in PY-CF-MS agar medium. Pure cultures weregrown in PY-CF-MS broth medium and lyophilized. Inthe preparation of cultures for lyophilization, all ma-

nipulations were carried out in the anaerobic chamber.Organisms were identified according to the proceduresof Holdeman et al. (16) with PY-CF agar and brothmedia instead of PY medium. We also referred toBergey's Manual ofDeterminative Bacteriology (7).Numbers of cellulolytic organisms were determined

by using the most-probable-number (MPN) technique(9) in PMC broth medium, because numbers estimatedwith PMC agar medium were erratic. Three tubes ofthe PMC broth medium were inoculated with 0.5 ml ofcecal contents serially diluted 10-fold. After severaltransfers of the highest dilution of MPN culturesshowing cellulose disappearance in PMC broth medi-um, cellulolytic organisms were isolated from theenrichments by first diluting (18) them in PMC agarmedium. Organisms forming clear zones were purifiedby subculturing them several times in PMC agar.

Cellulolytic organisms were lyophilized and identifiedas described above.

In vitro fermentation studies. Media used for in vitrofiber fermentation studies were prepared anaerobicallyunder 100% CO2 (18) and had the following composi-tion: inorganic salts and reducing agent, as for allmedia; a freshly prepared extract of intestinal-cecalcontents, 33%; and one of the following substrates:1.0% ethanol-extracted cabbage, 0.5% PMC, or no

substrate (control). For each substrate, 90 ml of medi-um was prepared in a 250-ml round-bottom flask. Themedium was not autoclaved. Ethanol-extracted cab-bage was prepared by a method similar to that of Ehleet al. (12a); the cabbage was soaked in 95% ethanol(four changes at approximately 1-h intervals), driedovernight, and then ground in a Wiley mill with a 20-mm mesh screen.To determine the extent of fiber degradation by

mixed cecal organisms in vitro, the ceca from four ratswere placed in the anaerobic chamber, and theircontents were pooled. The contents were weighed,and 2 volumes of dilution medium were added. Thismixture was thoroughly homogenized with a Virtis"45" homogenizer. Equal volumes of the suspensionwere added to duplicate fermentation vessels contain-ing the different fiber substrates. The average inocu-lum size for all experiments was 2.9% (wt/vol; range,

2.35 to 3.15%). Flasks were removed from the anaero-

bic chamber, the atmosphere was changed to 100%

C02, and the flasks were incubated in a 37°C waterbath for 48 h. Digestibility of cell wall fibers in thesuspensions was determined (12a).

Analysis of fermentation products. Hydrogen wasanalyzed at room temperature with a Perkin-Elmer154B gas chromatograph (The Perkin-Elmer Corp.,Norwalk, Conn.) with a silica gel column and N2 as thecarrier gas. Formic acid was analyzed by the methodof Lang and Lang (21). All other VFA were measuredwith a Varian model 3700 gas chromatograph (VarianAssociates, Inc., Palo Alto, Calif.) equipped with aflame ionization detector under the following condi-tions: stainless steel column (6 ft- by 0.125-in. outerdiameter; 0.093-in. inner diameter [ca. 2 m by 3.2 mm;2.4 mm]) packed with 15% FFAP chromosan acidwash; injector, 200°C; oven, 144°C; detector, 219°C;carrier gas, argon at a flow rate of 16 psig. D- and L-lactic (28) and succinic (24) acids were estimatedenzymatically.

RESULTSGut fermentation. Organic acids were found in

both the small intestines (jejuna) and ceca of rats(Table 2), indicating that fermentation occurs inthe gastrointestinal tracts of rats, as found previ-ously (12). The fermentation in the small intes-tine appeared to be homolactic, as significantamounts of lactic acid, but no VFA, were detect-ed. The fermentation in the cecum yielded largeamounts of acetic, propionic, and butyric acids;some lactic acid was also present, but in concen-trations lower than those found in the smallintestine.

Fiber degradation by mixed cecal flora. Todetermine whether cecal microflora are capableof degrading dietary fiber, cecal contents wereinoculated into anaerobic media containing PMCor cabbage. Both of these fiber sources werefermented by the mixed cecal population. Cab-bage fermentation was significantly greater (Ta-ble 3).

TABLE 3. Percent cabbage and PMC digested bymixed cecal flora after 48 h of incubation"

% of substrate digestedExpt

Cabbage Cellulose

1 68 36II 63 20

aInoculum: 2.9% (wt/vol) (range, 2.35 to 3.15%) ofcecal contents.

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TABLE 4. Total numbers of organisms andnumbers of each genus isolated from the intestinal

contents of ratsa

Organism No. of organisms(x108/g)Total ............. 8.68-56.9 (31)bLactobacillus spp.............8.45-56.9 (6)'Bifidobacterium spp ............0.07-5.29 (3)CEubacterium sp............ 0.23 (1)c

a Numbers shown are the ranges of six experiments.b Mean of six experiments.c Number of experiments in which organism was

isolated; six experiments (i.e., six animals) were done.

Enumeration and isolation of cellulolytic bacte-ria. After unsuccessful initial attempts (i.e.,clearing of cellulose was not observed) to enu-merate and isolate cellulose-degrading orga-nisms by using a PMC agar medium with acellulose concentration of 0.8% and an agarconcentration of 1.5%, we began using PMCbroth medium. The absence of agar in the medi-um allowed cellulose to be degraded. Initially,single tubes of PMC broth medium were inocu-lated with the serially diluted intestinal material.Results indicated that the number of cellulolyticbacteria per gram of contents (wet weight) wasapproximately five orders of magnitude lower inthe small intestine (approximately 103/g) than inthe cecum of rats (data not shown). According-ly, only the cellulolytic flora of the cecum wasinvestigated further.The MPN of cellulose-degrading organisms

was estimated from the number of inoculatedtubes in which cellulose degradation was detect-ed. Degradation was usually visible within 8days, but tubes showing no degradation wereincubated further (up to 3 months). Cellulolyticorganisms accounted for approximately 6.3% ofthe total number of viable bacteria in the cecumof rats. The number of viable organisms was 72x 108/g (average of six animals; range, 59 to102), and the number of cellulolytic organismswas 4.53 x 108/g (range, 0.81 to 12.2).

Isolation of cellulolytic bacteria from the cel-lulose enrichments was finally accomplished byfirst transferring the highest dilution of brothshowing cellulose degradation in PMC brothmedium several times. Cellulolytic organismswere isolated from the enrichments by firstdiluting them in a PMC agar medium that con-tained 0.5% PMC and only 0.5% agar. The lowerconcentration of agar allowed visible clearing tooccur. The lower cellulose concentration andmedium volume (3 ml) made it easier to see theclearings. After incubation for 16 to 20 days, twokinds of cellulose degradation zones were ob-served in the thin cellulose agar film; organismsresponsible for forming the zones were purified

by transferring them several times into PMCagar medium. The kind of clear zone that ap-peared most frequently (in material from 16 of 21animals) showed no visible colony. The orga-nisms isolated from this kind of clear zone weresmall, gram-negative rods and were classified asBacteroides succinogenes. The less frequentlyobserved clear zones contained a colony (in 5 of21 animals) that yielded gram-positive cocci inpairs or short chains. These organisms wereidentified as Ruminococcus flavefaciens. A de-scription of the characterization of these twotypes of cellulose-degrading organisms is pre-sented elsewhere (25).Because the use of PMC agar medium proved

so successful for isolating rat cecal cellulolyticorganisms from PMC broth cultures, attemptswere made to enumerate and isolate cellulolyticorganisms directly from cecal contents by usingthis same medium. Although discrete clearingswere detected and isolations were successfulwith PMC agar medium, numbers of cellulosedegraders, as estimated from the number ofclear zones, were erratic and slightly lower thanthose determined with the MPN technique. Inaddition, B. succinogenes strains were the onlycellulose degraders isolated from these agar dilu-tions. This was the case even when ruminococci(and not B. succinogenes) were isolated from theMPN broth inoculated with the same dilutedsample of cecal material. This may be becausethe ruminococci were present in such low num-bers compared to the non-cellulolytic organisms(non-cellulolytic bacteria outnumbered cellulo-lytic bacteria 17 to 1) that they simply could notcompete with the more numerous members ofthe flora that intercepted products of ruminococ-cus cellulase hydrolysis. Thus, it appears thatthe MPN technique is the most reliable approachto enumeration and isolation of cellulolytic orga-nisms.Enumeration and isolation of non-cellulolytic

bacteria. The numbers and kinds of predominantnon-cellulolytic organisms isolated from the in-testinal and cecal contents of rats are shown inTables 4 and 5. A total of 93 organisms wereisolated in six different experiments (41 and 52from intestine and cecum, respectively). Totalnumbers of viable organisms detected in thececa of rats were higher than those in the smallintestine. The predominant organisms isolatedfrom the small intestine were Lactobacillus spp.and Bifidobacterium spp. The most numerousorganisms detected in ceca were Lactobacillusspp., Eubacterium spp., Veillonella spp., andBacteroides sp.

DISCUSSIONThe purpose of the experiments described

here was to isolate one of the most important

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TABLE 5. Total numbers of organisms andnumbers of each genus isolated from the cecal

contents of ratsaNo. of organismsOrganism (x 108/g)

Total........... 29.2-208 (89.7)bLactobacillus spp........... 12.7-125 (6)'Eubacterium spp. ........ ... 7.34-65.4 (5)cBacteroides sp. ........ .... 6.29 (1)cEscherichia coli ........... 1.05 (1)'Streptococcus sp........... 2.13 (1)'Veillonella spp........... 2.62-17.8 (2)'

a Numbers shown are ranges of six experiments.b Mean of six experiments.c Number of experiments in which organism was

isolated; six experiments (i.e., six animals) were done.

groups of organisms involved in fiber fermenta-tion, the cellulolytic organisms, to determinewhether or not this dietary fiber is digested inthe ceca of rats, as has been suggested (35). Inrat ceca, cellulolytic bacteria were found in highnumbers (0.5 x 108 to 12.2 x 108/g) with theMPN technique and accounted for 6.3% of thetotal organisms isolated. The predominant cellu-lose degrader isolated was B. succinogenes.When human fecal material was examined byBryant and co-workers, also with the MPNtechnique and pebble-milled (PM) Whatman no.1 cellulose broth, a cellulolytic Bacteroides sp.was found in high numbers (greater than 108/g).The organism isolated, however, accounted foronly 0.3% of the total viable flora and was foundin only one of five individuals sampled (1, 2).Since Ehle found that all human fecal microbialpopulations tested were able to ferment bran(9.3% cellulose) and cabbage (31.1% cellulose)regardless of diet (12a), the major cellulolyticflora of humans most probably remains to beisolated. Perhaps the organisms could be foundif cabbage cellulose was used as a substrate.The numbers of cellulolytic organisms detect-

ed in the present study were significantly higherthan those found in ceca of other animals, suchas the guinea pig (approximately 107/g [11]),rabbit (0.05 x 107 to 7.2 x 107 [15]), and horse(0.007 x 106 to 1 x 106 [10]). Although B.succinogenes was the predominant cellulose-degrading organism isolated from rat ceca, Ru-minococcus sp. was the only type of cellulolyticbacteria isolated from guinea pig (11) and rabbit(15) ceca. Several types of cellulolytic bacteriahave been isolated from horse ceca (similar toRuminococcus sp., Bacteroides sp., Clostridiumsp., and Butyrivibrio sp. [10]), but none of thesehas been identified and characterized.We may have obtained higher numbers of

cecal cellulolytic organisms in our studies than

did previous workers because of the differentisolation methods used. Enumeration and isola-tion of cellulose degraders from guinea pigs,rabbits, and horses were accomplished by inocu-lating diluted cecal contents directly into a cellu-lose medium containing normal agar concentra-tions (i.e., by selective isolation). In each case,the cellulose source was different (guinea pig,PM Solka Floc [11]; rabbit, PM Whatman no. 1[15]; horse, a "fine suspension" [34] of cellulose[10]). In our experiments, selective isolation in acellulose (PM Whatman no. 1) agar mediumcontaining vitamins and VFA was not successfulunless the agar concentration (purified agar) waslowered to 0.5%. Even then, accurate enumera-tion was impossible because the results obtainedwere erratic. The most reliable method for esti-mating numbers of cellulolytic organisms wasthe MPN technique in PMC broth medium;isolation from broth was then accomplished inPMC agar medium with 0.5% agar. Consideringthe high numbers of organisms isolated in ourstudies and the inability of our B. succinogenesstrains to degrade cellulose in PMC agar mediumwhich contained a normal amount of agar, theconcentration of agar used in selective mediamay have prevented isolation of the predomi-nant cellulolytic bacteria from guinea pig, rabbit,and horse ceca.

This hypothesis is supported by the findingthat the use of high concentrations of agar withPM Whatman no. 1 cellulose also made growthof rumen strains of B. succinogenes difficult inrumen studies; this organisms was either notisolated (37, 38) or was isolated infrequently andin low numbers (17). The organism was onlyisolated selectively in significant numbers whenacid-treated PM cotton cellulose was included inan agar medium with a normal agar concentra-tion (13, 14, 19, 23, 33, 37). B. succinogenes wasalso isolated nonselectively along with otherpredominant rumen bacteria when a mediumdesigned to support growth of all rumen bacteriawas inoculated from dilutions of rumen fluid (3-6, 22). One of the cellulolytic B. succinogenesstrains isolated in this nonselective manner, S85(the neotype strain [8]), was unable to grow in amedium containing PM Whatman no. 1 cellulose(0.4 to 0.5%) and the normal concentration ofagar. When the agar concentration was lowered,however, S85 was able to grow and utilizecellulose in the presence of agar (25).

Clearly, further studies must be done to opti-mize cellulose agar media such that all cellulose-degrading gut anaerobes will be able to grow.This would assist in the accurate enumeration ofthese organisms from many different environ-ments.The results we obtained concerning non-cellu-

lolytic organisms isolated in this study are simi-


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lar to those reported previously (26, 27, 29). Theonly significant differences are that we detectedhigher numbers of lactobacilli (26, 27, 29) thandid other workers and slightly lower numbers ofBacteroides sp. than were detected in two stud-ies (26, 27). The reason for such differences isunclear, as adequate anaerobic techniques were

employed in all the studies. The results, howev-er, may have been influenced by different mediaused for enumeration and isolation of the orga-

nisms in each study (26, 27, 29).Since it is now clear that cellulose is degraded

by microbes from the ceca of rats, further stud-ies must be done to determine how the fermenta-tion of dietary fiber in the lower gut eitherdirectly or indirectly protects the animals fromcontracting diseases associated with diets low infiber (35).

ACKNOWLEDGMENTS

We thank R. L. Baldwin, Ann McGuire, and Irmelin Probstfor their critical reading of this manuscript and Carol Lane andCarole Pauls for technical assistance.

This study was supported by Public Health Service grantAM20872-03 from the National Institutes of Health.

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12a.Ehle, F. R., J. B. Robertson, and P. J. Van Soest. 1982.The influence of dietary fibers on fermentation in the largeintestine of humans. J. Nutr. 112:158-166.

13. Gilchrist, F. M. C. 1965. Influence of diet on functionalgroups of rumen bacteria fermenting carbohydrates andlactate, p. 559-564. In G. J. Snowball (ed.), Science andmedicine in Central Africa. Pergamon Press, Inc., Ox-ford.

14. Gilchrist, F. M. C., and A. Kistner. 1962. Bacteria of theovine rumen. I. The composition of the population on a

diet of poor teff hay. J. Agric. Sci. 59:77-83.15. Hall, E. R. 1952. Investigations on the microbiology of

cellulose utilization in domestic rabbits. J. Gen. Microbi-ol. 7:350-357.

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cellulolytic and non-cellulolytic bacteria in rat - applied and

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