A Simple Method (“Plate‐in‐Bottle Method”) for the Cultivation of Fastidious Anaerobes

Benno

2002

376

318

The human gut microbiota from three healthy subjects were compared by the use of a sequence analysis of 16S rDNA libraries and a culture‐based method. Direct counts ranged from 1.9 × 1011 to 4.0 × 1011 cells/g (wet weight), and plate counts totaled 6.6 × 1010 to 1.2 × 1011 CFU/g (wet weight). Sixty to seventy percent of the bacteria in the human intestinal tract cannot be cultured with currently available methods. The 16S rDNA libraries from three subjects were generated from total community DNA in the intestinal tract with universal primer sets. Randomly selected clones were partially sequenced. All purified colonies detected from the surface of the agar plate were used for a partial sequencing of 16S rDNA. On the basis of sequence similarities, the clones and colonies were classified into several clusters corresponding to the major phylum of the domain Bacteria. Among a total of 744 clones obtained, approximately 25% of them belonged to 31 known species. About 75% of the remaining clones were novel “phylotypes” (at least 98% similarity of clone sequence). The predominant intestinal microbial community consisted of 130 species or phylotypes according to the sequence data in this study. The 16S rDNA libraries and colonies included the Bacteroides group, Streptococcus group, Bifidobacterium group, and Clostridium rRNA clusters IV, IX, XIVa, and XVIII. Moreover, several previously uncharacterized and uncultured microorganisms were recognized in clone libraries and colonies. Our results also showed marked individual differences in the composition of intestinal microbiota.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Phylogenetic Analysis of the Human Gut Microbiota Using 16S rDNA Clone Libraries and Strictly Anaerobic Culture‐Based Methods

Benno

2002

376

318

“…A 0.5 g fecal sample was suspended in dilution buffer (10). Then, 50 µl samples of appropriate dilutions were plated anaerobically on medium 10 (3) using the "plate-in-bottle" method (20).…”

Section: Methodsmentioning

confidence: 99%

Fecal Microbial Diversity in a Strict Vegetarian as Determined by Molecular Analysis and Cultivation

Benno

2002

135

Fecal microbial diversity in a strictly vegetarian woman was determined by the 16S rDNA library method, terminal restriction fragment length polymorphism (T‐RFLP) analysis and a culture‐based method. The 16S rDNA library was generated from extracted fecal DNA, using bacteria‐specific primers. Randomly selected clones were partially sequenced. T‐RFLP analysis was performed using amplified 16S rDNA. The lengths of T‐RF were analyzed after digestion by HhaI and MspI. The cultivated bacterial isolates were used for partial sequencing of 16S rDNA. Among 183 clones obtained, approximately 29% of the clones belonged to 13 known species. About 71% of the remaining clones were novel “phylotypes” (at least 98% similarity of clone sequence). A total of 55 species or phylotypes were identified among the 16S rDNA library, while the cultivated isolates included 22 species or phylotypes. In addition, many new phylotypes were detected from the 16S rDNA library. The 16S rDNA library and isolates commonly included the Bacteroides group, Bifidobacterium group, and Clostridium rRNA clusters IV, XIVa, XVI and XVIII. T‐RFLP analysis revealed the major composition of the vegetarian gut microbiota were Clostridium rRNA subcluster XIVa and Clostridium rRNA cluster XVIII. The dominant feature of this strictly vegetarian gut microbiota was the detection of many Clostridium rRNA subcluster XIVa and C. ramosum (Clostridium rRNA cluster XVIII).

“…The total counts were expressed as number of bacteria cells per 1 g wet weight of cecal content. Other 0.05 ml samples of appropriate dilution were spread on anaerobically sterilized medium 10 (M10) (0.05% glucose, 0.05% cellobiose, 0.05% soluble starch, 3.75% minerals, 0.025% L-cysteine HCl · H 2 O, 0.0001% resazurin, 0.4% Na 2 CO 3 , 0.2% trypticase, 0.05% yeast extract, 0.31% volatile fatty acid, 0.001% hemin, and 2.0% agar, pH 6.8) (5) by the use of the plate-in-bottle method (16). After 3 days of incubation at 37 C, all visible colonies were carefully counted.…”

Section: Methodsmentioning

confidence: 99%

Phylogenetic Analysis of Cecal Microbiota in Chicken by the Use of 16S rDNA Clone Libraries

Lan

et al. 2002

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture‐based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate‐in‐bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C Gram‐positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C Gram‐positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.