Distribution of viable and non-viable bacteria in thegastrointestinal tract of gnotobiotic and conventionalratsBrigitta Kleessen, Jutta Noack and Michael Blaut

From the German Institute of Human Nutrition (DIFE), Potsdam-Rehbrucke, Germany

Correspondence to: Dr. Brigitta Kleessen German Institute of Human Nutrition Potsdam-Rehbrucke D-14558Bergholz-Rehbrucke Germany. Tel: +49 33200 88 440; Fax:+49 33200 88 407; E-mail: kleessen@www.dife.de

Microbial Ecology in Health and Disease 1999; 11: 218–225

To determine the proportion of viable and non-viable bacteria in the various sections of the gastrointestinal tract of rats, the contentsof jejunum, cecum, proximal and distal colon from conventional rats and rats monoassociated with Bacteroides thetaiotaomicron wereanalyzed with four different methods. The number of viable and non-viable bacteria were determined microscopically with thefluorescence-based BacLight staining technique (L/D) that distinguishes organisms with the nucleic acid-binding dyes Syto 9 andpropidium iodide. The viable bacteria were also enumerated by fluorescent in situ hybridization (FISH) with a mixture of oligonucleotideprobes targeting the 16S rRNA and by cultivation on agar plates. The number of total bacteria was determined microscopically after cellstaining with 4%,6-diamidino-2-phenylindol (DAPI). When applied to the contents of various sections of the rats’ gastrointestinal tract thestaining with L/D and FISH yielded similar cell counts. The highest percentage of viable bacteria, determined as the ratio of L/D-stainedto total counts, was detected in the cecum of both rat models (77–87%). Only 50% of the DAPI-stained cells in the colonic contents ofconventional rats were also detected by FISH or the viability stain. In gnotobiotic rats 70% of the cecal or colonic cells detected withDAPI were recovered by plating. In conventional rats this proportion decreased to 25%. In conclusion, (I) the culture-based methodprovides an incomplete picture of the viable population in conventional rats and (II) in situ methods such as L/D and FISH are an usefulalternative to cultivation on agar plates for measuring bacterial viability in the rats’ gut. Key words : intestinal microflora, conventionalrat, gnotobiotic rat, in situ hybridization, viability staining.

ORIGINAL ARTICLE

INTRODUCTION

The large intestine of humans and animals is colonized bya vast variety of bacteria representing a broad range ofphysiological properties (1). At least 400 to 500 differentbacterial species are thought to be present in the intestine,and up to 1012 bacteria are found in every gram of feces.In order to fully explore the importance of the intestinalpopulations to the host and to characterize factors thatcan affect this community, we have to know exactly thecomposition of the intestinal flora. Our present knowledgeof the composition and activity of the intestinal microflorais mainly based on information obtained from analyses offeces. However, pronounced regional differences exist inthe gastrointestinal (GI) tract with respect to the metabolicactivities of the microflora (2). Therefore, more basicinformation on the proportion of viable and/or activebacteria in the various sections of the GI tract is needed.Traditional cultivation methods such as plating or most-probable number techniques do not provide appropriatetools because it is now well recognized that only a minorfraction of the intestinal bacteria is accessible by tradi-tional cultivation methods (3). Fluorochrome stains such

as acridine orange or 4%6-Diamidino-2 phenylindol (DAPI)allow the microscopic enumeration of total bacteria intheir natural habitat (4). Furthermore, fluorescence-basedapproaches have been developed to evaluate bacterial vi-ability (5–7). Besides methods that detect metabolic activi-ties, redox potential, or membrane integrity, new nucleicacid-specific blue-excitable dyes such as SYBR-II andSyto-9 have been developed (8). Recently, a new systemfor assessing bacterial cell viability, the LIVE/DEAD®

BacLight Bacterial Viability Kit (L/D), has become com-mercially available (9). The kit provides an appropriatemixture of the nucleic acid stains SYTO 9 and propidiumiodide that confer to bacteria with an intact cytoplasmaticplasma membrane a green fluorescence and to bacteriawith a damaged cytoplasmatic membrane a red fluores-cence. The excitation/emission maxima are approximately480 nm/500 nm for SYTO 9 and 490 nm/635 nm forpropidium iodide. SYTO 9 is permeable to virtually all cellmembranes. Propidium iodide is generally excluded fromviable cells. Furthermore, fluorescence-labeled probes suchas antibodies (10) and rRNA-targeted oligonucleotideshave been introduced in microbiology (11–13). Theseprobes allow the in situ identification of intestinal bacteria

© Scandinavian University Press 1999. ISSN 0891-060X Microbial Ecology in Health and Disease

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Enumeration of intestinal bacteria 219

without the necessity to grow the organisms. The applica-tion of fluorescence in situ hybridization (FISH) withrRNA-targeted oligonucleotide probes in intestinal sam-ples may be limited by low ribosomal content or restrictedcell permeability (14). Therefore, the sensitivity of whole-cell hybridization must be improved. To identify bacterialcells with high or low rRNA content in the various sec-tions of the GI tract we have hybridized with a mixture offive Bacteria-directed probes (EUB mix).

In this investigation four methods were employed toenumerate viable and non-viable bacteria in contents col-lected from the jejunum, cecum, proximal and distal colonof rats. Conventional and gnotobiotic rats were used forthese investigations because difficulties are encountered toget samples from the GI tract of healthy humans. Thenumber of viable and non-viable bacteria detected withL/D were related to the total bacterial counts determinedwith DAPI and compared with the number of viablebacteria enumerated either by FISH with EUB mix or bycultivation on agar plates and enumeration of colonies.Using this strategy, we were able to determine the propor-tion of viable and non-viable bacteria in the various sec-tions of the GI tract of rats.

MATERIALS AND METHODS

Two sources of microorganisms were used in this study,intestinal samples of rats and pure cultures of intestinalbacteria.

Animals and intestinal samples

Intestinal samples were obtained from 6 conventional(Tierzucht Schonwalde GmbH, Schonwalde, Germany)and 6 gnotobiotic (Inzucht, DIfE, Bergholz-Rehbrucke)male Wistar rats. The latter rats were monoassociated withBacteroides thetaiotaomicron (DSM 2079) (inoculum 1.2×1011 living cells). Rats with an initial body mass of 165915 g were individually placed in macrolon cages andhoused in a room with a 12-h light: dark cycle and at atemperature of 2292°C. The cages of the gnotobiotic ratswere arranged in sterile isolators equipped with a sterilewater supply. All rats were allowed to consume a standarddiet (Altromin 1320) and water ad libitum for 3 weeks. Atthe end of the experiment the rats were killed by decapita-tion. The abdomen of the rats was opened surgically, theintestine was ligated and transported within 5 min to ananaerobic chamber (MK3 anaerobic workstation, dW Sci-entific, West Yorkshire, England) under 85 % N2, 10 % H2

and 5 % CO2 for processing. Portions of 0.5 g werecollected anaerobically from the contents of distal je-junum, cecum, proximal and distal colon. Samples weresuspended evenly in 4.5 ml filter-sterilized deionized H2Oprior to fixation for hybridization, staining or culturalenumeration as described below.

Bacterial cultures, culture medium, and growth conditions

Reference strains used for validation of the LIVE/DEAD®

BacLight (L/D) microscopic technique (Table I) were ob-tained from the American Type Culture Collection(ATCC, Rockville, MD) or from the German Collectionof Microorganisms and Cell Cultures (DSMZ, Braun-schweig, Germany). Fusobacterium 6arium (ATCC 8501),Bacteroides thetaiotaomicron (DSM 2079), Bifidobacteriumlongum (ATCC 15707) and Eubacterium 6entriosum(ATCC 27560) were also used in this study for measuringthe fluorescence intensity of bacterial cells in variousgrowth phases after FISH as indicated below. For growthexperiments strains were cultured in prereduced Schaedleranaerobic broth (SB) supplemented with vitamin K andhemin (OXOID, Unipath GmbH, Wesel, Germany) underanaerobic conditions at 37°C. After overnight incubation 1ml (10 ml) was transfered to 9 ml (90 ml) SB and incu-bated at 37°C. Growth of strains was monitored by mea-suring the change in turbidity at 600 nm with a

Table I

Staining of li6ing intestinal organisms with the LIVE/DEAD Ba-cLight Kit

Staining proper-Strain Originties percentagea

100DSM 935TClostridium coccoides94DSM 2151TBacteroides fragilis

ATCC 8501Fusobacterium 6arium 85Fusobacterium nucleatum DSM 20482T 83Veillonella par6ula DSM 2008T 77Bacteroides thetaiotaomicron DSM 2079T 74Escherichia coli DSM 498 72Pre6otella melaninogenica 71ATCC 25845Bacteroides 6ulgatus DSM 1447T 70Acidaminococcus fermentans DSM 20731T 65Eubacterium 6entriosum ATCC 27560 61Clostridium butyricum 60DSM 552T

Bifidobacterium longum 58ATCC 1570756DSM 20470TPeptostreptococcus magnus

Bifidobacterium infantis DSM 20088T 54Lactobacillus acidophilus DSM 20079T 54

53DSM 20052TLactobacillus fermentumATCC 27340 53Ruminococcus productus

Eubacterium rectale ATCC 33656 51DSM 20174T 49Lactobacillus plantarum

Clostridium perfringens DSM 756T 47Eubacterium aerofaciens DSM 3979T 47

47DSM 20016TLactobacillus reuteriEnterococcus faecalis 42DSM 20478T

Enterococcus faecium DSM 20477T 38

a Average fluorescence intensity of the organisms relative to themaximal value obtained with Clostridium coccoides, arbitrarily setto 100%. Between 450 and 620 bacterial cells taken from expo-nentially growing cultures were analyzed per filter. The standarddeviation determined for triplicate samples was less than 4% ineach case.

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spectrophotometer (Beckman, Munich, Germany). For L/Dcells in the exponential growth phase (OD600 between 0.6and 0.9, usually 108–109 bacteria/ml) were harvested bycentrifugation (4 min, 5000×g) and carefully suspendedin 1 ml filter-sterilized (0.2 mm Minisart, Sartorius, Ger-many) deionized H2O. Samples for FISH were taken at 2h-intervals for 36 h and fixed as described below.

Fixation

For fluorescent in situ hybridization a 1-ml aliquot of eachintestinal sample or bacterial culture was fixed with 3volumes of fresh 4% (w/v) paraformaldehyde in phos-phate-buffered saline (PBS: 130 mM sodium chloride, 10mM sodium phoshpate buffer, pH 7.4) and incubated at4°C for 3 h. Cells were subsequently pelleted by centrifuga-tion in an Eppendorf microcentrifuge (5000×g, 4 min)washed with PBS, and resuspended in a 1:1 mixture ofPBS and 96 % (v/v) ethanol. The fixed samples were storedat −20°C and processed within 8 days.

DAPI staining

DAPI staining was used for the enumeration of totalbacteria. For each sample 1.0-ml aliquots of serially de-luted samples were incubated with DAPI (Sigma-AldrichGmbH, Deisenhofen, Germany) at a final concentration of2.0 mg ml−1 for 15 min in the dark.

Viable staining

Following the manufacturers’ instructions of the LIVE/DEAD® BacLight Bacterial Viability Kit (L/D), pur-chased from Molecular Probes Europe BV, Leiden,Netherlands, equal volumes of components A and B weremixed (9). Three microliters of this mixture was added to1.0 ml of the serially diluted bacterial suspension, followedby mixing and incubation in the dark for 25 min to achievebest results. The kit was validated with 25 intestinal refer-ence strains (Table I).

FISH

In situ hybridization was performed essentially as de-scribed by Manz et al. (15). An equimolar mixture of 5Bacteria-directed probes (EUB 338, EUB 785, EUB 927,EUB 1055, EUB 1088) (12), referred here to as EUB mix,was used to detect all bacteria in the fixed samples. Theoligonucleotides were purchased 5% labeled with the indo-carbocyanine dye Cy3 (Interactiva Biotechnologie GmbH,Ulm, Germany). Fixed cells were hybridized for 16 h at46°C in 50 m l of hybridization buffer (0.9 M sodiumchloride, 0.01 % sodium deodecyl sulfate [SDS] and 10mM Tris-HCl, pH 7.4) containing 5 ng of EUB mix per m l.Hybridized cell suspensions were washed at 48°C for 20min in hybridization buffer without SDS. The fluorescingcells were viewed as described below.

Filtration

Stained samples were filtered within 10 min through ablack polycarbonate membrane (0.2 mm pore size, Mil-lipore) placed on a nitrocellulose support membrane (0.45mm pore size) of a vacuum filtration unit (Schleicher andSchuell, Dassel, Germany) which was filled with 3 mlfilter-sterilized deionized water. The support membranewas used to promote uniform distribution of the bacteriaon the filter surface. All manipulations were conducted inthe dark. Following microfiltration filters were mountedon a glass microscope slide with the Slow Fade™ AntifadeKit (Molecular Probes Europe BV, Leiden, Netherlands)for microscopical enumeration.

Microscopy and image analysis

Hybridized and stained cells were examined with a Op-tiphot-2 epifluorescence microscope (Nikon, Dusseldorf,Germany) equipped with an 100 ×/1.30 Plan NeofluorPh4 oil immersions objective, a HBO 100 w/2 mecurylamp, and the filter blocks UV-2A (DAPI), G-2A (FISH:CY3), B-1H or the two-dye filterset Fluorescein/rho-damine (L/D: Syto 9/ propidium iodide). All samples wereenumerated in triplicate using a net micrometer. Generally,25 fields per slide were counted with 65–150 cells per field.The bacterial concentration per ml sample was calculatedas follows:

N=M×CF×DF

where M is the microscopic count, representing the aver-age number of bacteria per field, CF is the membraneconversion factor, calculated as the ratio of the effectivedrainage area (346 mm2) to microscopic field area (0.01513mm2) i. e. about 22,870, and DF is the dilution factor.

Using the L/D technique, cells fluorescing green wereconsidered viable and cells fluorescing red were considerednon-viable. Bacteria fluorescing in other colors such asyellow were rarely observed (B104 cells/g) and counted asnon-viable. To establish the basic protocol of the L/D kitand confirm its staining behavior on intestinal referencestrains (Table I) the fluorescence signal from viable stainedcells was quantified with the Image Software KS400 Re-lease 3.0 (Carl Zeiss). Samples were analyzed using a CarlZeiss Axioplan epifluorescence microscope. Digital imageswere captured with a 12-bit cooled CCD camera (Sensicam370KL). The average fluorescence intensity of the organ-ism with the highest fluorescence intensity was arbitrarilyset to 100%, and all other measurements related to thisvalue.

The fluorescence signals of hybridized cells in variousgrowth phases were analyzed by the same image analysisprogram (Table II). The mean fluorescence intensity percell taken from different growth phases was calculatedrelative to the maximum value obtained with mid-expo-nential-phase cells of Fusobacterium 6arium, arbitrarily setto 100%.

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Table II

Fluorescence intensity of intestinal bacteria after FISH with probe EUB mix in 6arious growth phases

Relative mean fluorescence intensity per cellbOriginStraina

Growth Phase StationaryStationaryLate-exponentialMid-exponential 5h 10h

100 95Fusobacterium 6arium 93ATCC 8501 84DSM 2079 83 82 75 72Bacteroides thetaiotaomicronATCC 15707 72 70 66 65Bifidobacterium longum

64 68 65ATCC 27560 60Eubacterium 6entriosum

a Strains were cultured in prereduced Schaedler anaerobic broth under anaerobic conditions at 37°C.b The mean fluorescence intensity per cell in different growth phases was calculated relative to the maximum value obtainedwith mid-exponential-phase cells of Fusobacterium 6arium, arbitrarily set to 100%. Between 180 and 320 bacteria wereanalyzed per sample. The standard deviation determined for triplicate samples was less than 3% in each case.

Plate counts

Culturability was determined by standard plate technique(16). After homogenization, the specimens were subjectedto a series of 10-fold dilutions (10−2–10−8) in a prere-duced saline buffer, and duplicate samples of 0.05 mlfrom each dilution were plated on Columbia blood agar(BioMerieux, Nurtingen, Germany). All manipulations ofthe anaerobic media were performed in an anaerobicchamber (MK3 anaerobic workstation, dW Scientific,West Yorkshire, England).

Dry weights

Gut contents were freeze-dried to constant weight undervacuum.

Statistical procedure

Results are expressed as the number of bacteria per mlor per g dry weight of the intestinal sample. Student’st-test was used in the statistical evaluation (SYSTAT,version 5.2., Systat, Inc., Chicago, IL), and values ofPB0.05 were regarded as significant.

RESULTS

Staining of intestinal bacteria with L/D

To test the applicability of L/D to intestinal bacteria 25representative intestinal species were grown to the expo-nential growth phase, and the fluorescence intensity ofthe viable cells was quantified after staining (Table I).Fluorescing cells could be easily distinguished from thedark background and readily quantified. The fluores-cence intensity varied considerably between the speciesinvestigated. Clostridium coccoides, Bacteroides, Fusobac-terium, Veillonella, Escherichia coli, Pre6otella exhibitedhigher fluorescence intensity than for instance Eubac-terium, Bifidobacterium, and Lactobacillus. Enterococcusdisplayed the lowest fluorescence intensity of all bacteria

investigated. The relationship between growth and thenumber of viable cells was investigated in a growth ex-periment with Bacteroides thetaiotaomicron (DSM 2079).Samples were taken at 1, 4, 9 and 24 h after start of theexperiment (Fig. 1). Plate counts and microscopic countsof L/D-stained cells were similar at 1, 4, and 9 h. At 24h, the plate counts were slightly higher than the micro-scopic cell counts. In contrast to pure-culture samples,intestinal samples displayed unspecific fluorescence ofparticles or debris. However, these could be clearly dis-tinguished from fluorescing cells and did therefore notaffect cell counting. Although the viable cells stainedwith L/D exhibited different levels of green fluorescenceintensity, these cells could be clearly distinguished fromthe non-viable cells exhibiting red fluorescence.

Growth-related changes in fluorescence intensity ofintestinal bacteria after FISH with EUB mix

The fluorescence intensity of four representative intesti-nal species in various growth phases after FISH withEUB mix was determined (Table II). All strains grewwell in SB medium. The generation times for Bacteroidesthetaiotaomicron, Eubacterium 6entriosum, Fusobacterium6arium, and Bifidobacterium longum were 61, 66, 77, and79 min, corresponding to growth rates of 0.98, 0.91,0.78, and 0.76 doubling per h, respectively. The meanfluorescence intensity of cells in the exponential growthphase decreased in the following sequence: Fusobacter-ium 6arium\Bacteroides thetaiotaomicron\Bifidobacteriumlongum\Eubacterium 6entriosum. A decrease in the hy-bridization signal intensity was observed in stationary-phase cells as compared to mid-exponential-phase cells.The flourescence signal decreased faster in Fusobacterium6arium (by 16%), than in Bacteroides thetaiotaomicron(by 13%), Bifidobacterium longum (by 10%), and Eubac-terium 6entriosum (by 6%).

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Distribution of bacteria in the GI tract of gnotobiotic andcon6entional rats

The microbial population in the various segments of theGI tract of gnotobiotic and conventional rats was char-acterized by several enumeration methods (Tables III–IV). Whereas the jejunum contained few organisms, adense population of bacteria was evident in the cecumand colon. The highest percentage of viable bacteria, de-termined as the ratio of L/D-stained to total DAPIcounts was found in the cecum of the rats. Total cells incecum and colon were higher in conventional rats thanin rats monoassociated with Bacteroides thetaiotaomicron(1.37×1011 vs. 6.94×1010 cells/g dry weight). Bacterialcell counts in the contents of the various sections of thegnotobiotic rats’ GI tract were very similar no matterwhether enumeration was done with FISH, LD or plat-ing (Table III). The counts in the jejunum determinedwith these methods was 54 % of those determined withDAPI staining. Cecal and colonic contents of gnotobi-otic rats contained higher proportions of viable bacteriathan jejunal contents (66–87 %). Analysis of intestinalsamples from conventional rats with L/D and FISHyielded essentially the same results (Table IV). However,only 56% and 53% of the total cells in colonic contentswere also detected by FISH and L/D, respectively. Incontrast, less than 25 % of the bacteria detected withDAPI in the cecum or colon could be recovered fromagar plates (PB0.05). The observed interindividual vari-ations were independent of the method used. The resultsobtained for samples from gnotobiotic rats analyzed byFISH (0.12) and for samples from conventional ratsstained with DAPI (0.11) had the lowest coefficient ofvariance.

DISCUSSION

This study reveals that the majority of the cecal bacteriain conventional rats and rats monoassociated with Bac-

teroides thetaiotaomicron is viable, but that 75 % of thetotal bacteria in conventional rats and 30% of B.thetaiotaomicron in the gnotobiotic rats elude cultivation(Tables III–IV). The low recovery of bacteria from nat-ural habitats on growth media has been observed previ-ously (12, 15). Therefore, it has been suggested that theinability of bacteria to form colonies might be due totheir inability to grow on the media used. Alternativelyit has been suggested that such bacteria may have en-tered a viable but nonculturable state (17). However, itis reasonable to assume that most organisms could becultivated if only the exact growth conditions wereknown.

It would be expected that the number of bacteria im-permeable to propidium iodide, i.e. cells with an intactcytoplasmatic membrane, exceeds the number of countsdetermined by plating. This applies in particular to thedistal parts of the colon, in which the substrateavailability for fermentation is limited (18) and bacteriamay grow at a low rate or be in a dormant state (19).The results obtained for conventional rats are in accor-dance with this notion. In contrast to the situation inconventional rats, the cecal and colonic cell counts ingnotobiotic rats determined with L/D did not differ fromthose determined with plating. This indicates that theintestinal contents of germfree rats represent a suitablegrowth medium for Bacteroides thetaiotaomicron.

It is worthwhile to note that the analysis of intestinalcontents by the viability stain (L/D) and FISH gave sim-ilar results. This means that the bacterial cells in theintestine with an intact cytoplasmatic membrane containsufficiently high numbers of 16S rRNA copies to resultin a clearly detectable fluorescence signal. Moreover, theuse of Cy3 as fluorescent dye and a mixture of fiveprobes specific for the domain Bacteria, each of whichtargets a different sites on the 16S rRNA molecule, haveconsiderably increased the sensitivity of the whole cell

Fig. 1. Counts of Bacteroides thetaiotaomi-cron (DSM 2079) during growth onSchaedler anaerobic broth determined byenumeration of colonies after plating onColumbia blood agar or by microscopicenumeration after staining with the LIVE/DEAD BacLight Kit. Standard deviationswere determined from triplicate samples.

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Table III

Comparison of bacterial densities in the intestinal tract of gnotobiotic rats counted by different methodsa

Viable population Viable populationIntestine Non-viable population Cultural populationTotal organismsFISH with LIVE/DEADDAPI LIVE/DEAD Columbia agar

BacLight KitEUB mix BacLight Kit

% Counts % Counts % CountsCountsb % Counts %

7.48*108 100 4.03*108 54 4.18*108 56 3.28*108Jejunum 44 3.85*108 51(distal)

100 5.47*1010 82 5.86*1010 87 1.03*1010Cecum 15 4.75*1010 716.71*1010

6.91*1010 100 5.05*1010 73 4.79*1010 69Colon 2.15*1010 31 4.93*1010 71(proximal)

7.20*1010 100 4.89*1010 68 4.74*1010 66 2.67*1010 37 4.94*1010Colon 69(distal)

0.12 0.19c.v.c 0.22 0.260.23

a Rats were monoassociated with Bacteroides thetaiotaomicron (DSM 2079).b Data are counts/gram of dry weight and represent means from 6 rats.c Coefficient of variance pooled for each separate method from the different intestinal part.

detection method used by us: The fluorescence signals ofthe target bacteria were up to 4 times more intense thanthose of standard FISH with the fluorescein isothio-cyanate-labeled probe EUB338 (data not shown).

The data obtained with FISH and L/D do not provideany information on the abundance of fast, slow, or non-growing organisms in the complex intestinal environ-ment. However, one should take into account that theribosomal content is proportional to the growth rate ofa culture (14, 20). Changes in nucleic acid compositionor structure may also occur during starvation (21, 22).The cellular rRNA content and the growth rate havebeen correlated in order to study the in 6i6o growth ratedirectly from fixed cells of Escherichia coli BJ4 coloniz-ing the large intestine of streptomycin-treated mice byFISH with rRNA targeted probes (23). The authorsdemonstated that E. coli BJ4 differentiates duringgrowth in the intestine into two distinct populations, onethat grows very fast and one that multiplies more slowly(24).

The above arguments prompted us to determine thefluorescence intensity of four intestinal bacterial speciestaken from pure cultures at various growth phases afterFISH (Table II). Although the organisms grew on SB atsimilar rates (generation times between 61 and 79 min),the fluorescence intensity of the cells varied considerablyin the logarithmic growth phase. Moreover, in case ofFusobacterium 6arium the fluorescence signal decreased toabout 84% in the stationary phase. This may be due todifferences in the concentration of target rRNA or to analtered permeability of cells to the probes (25). It isinteresting to note that our results are consistent withthe findings of Langendijk et al. (26), who observed dif-ferent levels of fluorescence intensity in a number of

species when the universal probe Uni 519 was used forFISH.

In our study, the proportion of viable and non-viablebacteria in the various sections of the GI tract varieddepending on the rat models used (Tables III–IV). Thefinding that the proportion of cells detected with FISHor L/D was generally lower in colonic contents of con-ventional rats than in colonic contents of gnotobioticrats (54% vs. 69%) may be attributed to a higher celldensity (1.38×1011 vs. 7.05×1010) and a reduced sub-strate availability in the former rats (27).

The high number of bacteria detected by L/D andFISH in the cecum of both rat models reflects a consid-erable fermentation capacity in the cecum. In contrast tothe cecum, the more distal regions of the large intestineare characterized by a steady decrease in fermentablesubstrates (18). It is therefore not surprising that theproportion of viable bacteria in the colon of conven-tional rats is lower than in their cecum.

In view of the assumed differences in the substrateavailability in the various parts of the GI tract of con-ventional or monoassociated rats, more information onthe actual in 6i6o growth rate of the organisms areneeded. We are currently exploring the possibility to esti-mate the growth rate of bacterial species in the intestinewith FISH, as recommended recently by Poulsen et al.(23).

Taken together, this is the first study that has ana-lyzed systematically the proportion of viable and non-vi-able bacteria in the various parts of the GI tract of rats.Further studies are needed to characterize the representa-tive species that make up the predominant populationgroups in the GI tract. Keeping in mind the bias causedby cultivation, such studies should rely on in situ iden-tification of individual cells with nucleic acid probes.

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Table IV

Comparison of bacterial densities in the intestinal tract of con6entional rats counted by different methods

Intestine Total organisms Viable population Viable population Non-viable population Cultural populationFISH with LIVE/DEADDAPI LIVE/DEAD Columbia agar

BacLight Kit BacLight KitEUB mix

% Counts % Counts % Counts % Counts %Countsa

100 2.18*108 43Jejunum (distal) 2.35*108 47 2.54*108 51 1.71*108 345.03*108

100 9.89*1010 72 1.06*1011 771.38*1011 3.35*1010Cecum 24 3.16*1010 23100 7.51*1010 56 7.10*1010 53 6.55*1010Colon (proximal) 49 2.42*1010 181.35*1011

100 7.66*1010 55 7.45*1010 531.40*1011 6.10*1010 44 2.87*1010 20Colon (distal)0.18 0.23 0.16c.v.b 0.280.11

a Data are counts/gram of dry weight and represent means from 6 rats.b Coefficient of variance pooled for each separate method from the different intestinal parts.

ACKNOWLEDGEMENTS

This work was supported by the European Research ProjectFAIR-CT-97-3035. The authors would like to thank LudgerHartmann, Renate Herzog, Susanne Dietrich, and Ines Grunerfor providing and caring the gnotobiotic rats. The expert techni-cal assistance of Sabine Schmidt is acknowledged.

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