Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the speciesBacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and theClostridium lituseburense groups. Another probe was designed for the genera Streptococcus andLactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectalegroup. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 1010 cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectalegroup-specific probe detected a mean of 7.2 × 1010cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, andStreptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 107 to 7 × 108 per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.

Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.

No abstract

Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 104 or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60%o and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 104 cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 109 to 1.07 x 109 cells per liter. The mean cell volume varied between 0.042 and 0.074 ,um3, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria.

Oligotrophy, or the inability of bacterial cells to propagate at elevated nutrient concentrations, is a controversial phenomenon in microbiology. The exact cause of the unculturability of many indigenous marine bacteria on standard laboratory media has still not been resolved. Unfortunately, the physiology of such cells is difficult to investigate as long as high cell density cultures cannot be obtained. An extensive evaluation of experiments relating to oligotrophy and the cultivation of marine bacteria is presented in this review. When incorporating the findings of studies performed with molecular biological methods, the picture emerges that indigenous marine bacteria can be cultivated under certain cond~tions and that the 'ohgotrophic way of life' is a transient characteristic. Although strong generalisations should not be made w~t h respect to a biological system as diverse as the world's oceans, it should be anticipated that cells with unique physiological characteristics appear to exist in the oceanic system. When combining conventional physiological approaches with molecular biological techniques it is feasible to unveil the phenotypes that go with the encountered genotypes. In view of the enormous complexity of the oceanic system this will prove an ambitious, yet resourceful undertaking.

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.The human gastrointestinal (GI) tract harbors a diverse microbial community, which has important metabolic and protective functions in the GI tract (for a review see reference 18). Recent studies have indicated that interactions between the host and the bacterial community are of considerable importance but are very complex and are just starting to be understood (4,9,10,25). Most information about the bacterial community in the human GI tract has been obtained by selective cultivation of microbes from fecal samples. In the past 5 years, culture-independent approaches in which the sequence variability of the 16S rRNA genes has been used have demonstrated that most of the predominant bacteria in human fecal samples have not yet been obtained in culture, illustrating the limitation in our knowledge of these predominant organisms (16,23,24). In addition, denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis of fecal 16S ribosomal DNA (rDNA) and rRNA amplicons have been demonstrated to be powerful culture-independent approaches for determining and monitoring the bacterial community in feces (24,25). Such studies have revealed that the predominant bacterial community in human feces is relatively stable over time, is host specific, and is not significantly altered following consumption of certain probio...

Seven Gram-negative strains, isolated in 1990 from a 10 6 -fold dilution series of seawater from Resurrection Bay, a deep fjord of the Gulf of Alaska, were identified in a polyphasic taxonomic study. Analysis of 16S rDNA sequences and DNA-homology studies confirmed the phylogenetic position of all strains in the genus Sphingomonas and further indicated that all of the strains constitute a single homogeneous genomic species, distinct from all validly described Sphingomonas species. The ability to differentiate the species, both phenotypically and chemotaxonomically, from its nearest neighbours justifies the proposal of a new species name, Sphingomonas alaskensis sp. nov., for this taxon. Strain LMG 18877 T (l RB2256 T l DSM 13593 T ) was selected as the type strain.Keywords : Sphingomonas alaskensis sp. nov., identification, polyphasic taxonomy, marine ultramicrobacterium INTRODUCTIONMicrobiologists have been intrigued by the phenomenon of ' unculturability ' for over half a century, especially with respect to bacteria in the open ocean (MacLeod, 1985). This notion has now virtually reached the status of a dogma but is still based primarily on the common observation that often numerically dominant marine bacteria cannot be grown on agar surfaces (Austin, 1988 ;Jannasch & Jones, 1959 ;Kogure et al., 1979Kogure et al., , 1980Poindexter & Leadbetter, 1986 ;Roszak & Colwell, 1987 ; Schut et al., 1997a, b ; Van Es & MeyerReil, 1982). These studies all point in the same direction and indicate that by using agar-plate counts and isolation procedures usually less than 0n1 % of the total community that is observed by direct microscopy can be cultured. As a logical consequence of this situation, most of our knowledge on the physiological properties of marine bacteria is based on those species that are Abbreviations : FAS, filtered-autoclaved seawater ; FAMEs, fatty acid methyl esters.The EMBL accession numbers for the sequences determined in this work are AF145752 (1280 bp), AF145753 (1416 bp) and AF145754 (1428 bp), respectively.readily obtained in culture but which mostly belong to a minority of the total community. Culture-independent molecular techniques are now widely used to obtain a thorough understanding of the identity and nature of the bacteria comprising marine heterotrophic communities, because of their apparent unculturability. This approach has indeed indicated the existence of numerous possibly ' new ' and unusual uncultured bacteria (Amann et al., 1995 ;Britschgi & Giovannoni, 1991 ;Fuhrman et al., 1992Fuhrman et al., , 1993Giovannoni et al., 1990Giovannoni et al., , 1995Ho$ fle & Brettar, 1996 ;Mullins et al., 1995 ;Schmidt et al., 1991). The developments in the use of molecular probes for the detection of natural bacterial populations and for the analysis of genetic diversity within communities have revolutionized our approach in microbial ecology so much that they may create the impression that isolation and cultivation of naturally occurring bacterial strains might soon cease to be required. However, th...