scholarly journals Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes

2002 ◽  
Vol 68 (9) ◽  
pp. 4225-4232 ◽  
Author(s):  
Erwin G. Zoetendal ◽  
Kaouther Ben-Amor ◽  
Hermie J. M. Harmsen ◽  
Frits Schut ◽  
Antoon D. L. Akkermans ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Specific Effect ◽  
Human Intestine ◽  
Fecal Samples ◽  
Flow Cytometric ◽  
Significant Difference

ABSTRACT 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.

scholarly journals Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ

1999 ◽  
Vol 65 (4) ◽  
pp. 1746-1752 ◽  
Author(s):  
Cleber C. Ouverney ◽  
Jed A. Fuhrman
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Microbial Communities ◽  
Fluorescent In Situ Hybridization ◽  
Amino Acid Uptake ◽  
Cell Types ◽  
Black Box ◽  
Acid Uptake ◽  
Substrate Uptake

ABSTRACT We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class Proteobacteria(α-Proteobacteria) and of the Cytophaga-Flavobacteriumgroup obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (∼60% of the universal probe-labeled cells and ∼50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.

Novel Nitrospira-like bacteria as dominant nitrite-oxidizers in biofilms from wastewater treatment plants: diversity and in situ physiology

2000 ◽  
Vol 41 (4-5) ◽  
pp. 85-90 ◽  
Author(s):  
H. Daims ◽  
P.H. Nielsen ◽  
J.L. Nielsen ◽  
S. Juretschko ◽  
M. Wagner
Keyword(s):  
Wastewater Treatment ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Wastewater Treatment Plants ◽  
Combined Application ◽  
Fundamental Understanding ◽  
Unculturable Bacteria ◽  
Rrna Sequences

The frequency and distribution of putatively nitrite-oxidizing, Nitrospira- like bacteria in nitrifying biofilms from two reactors receiving wastewater with different ammonia and salt concentrations were observed by fluorescent in situ hybridization. For this purpose, new 16S rRNA-directed oligonucleotide probes targeting the bacterial phylum Nitrospira and the three main lineages within this phylum were developed and evaluated. The diversity of Nitrospira-like bacteria in the reactors was additionally investigated by retrieval and comparative analysis of full 16S rRNA sequences from the biofilms. We found that, despite of the differences in the influent composition, Nitrospira-like bacteria form dominant populations in both reactors. In addition, first insights into the physiology of these still unculturable bacteria were obtained by the incubation of active biofilm samples with radioactively labeled substrates followed by the combined application of fluorescent in situ hybridization and microautoradiography. The results are discussed in consideration of the frequently observed dominance of Nitrospira-like bacteria in nitrifying bioreactors. Consequently, high priority should be assigned to future studies on the ecology and physiology of these organisms in order to increase our fundamental understanding of nitrogen cycling and to enable knowledge-driven future improvements of nitrifying wastewater treatment plants.

Immunoglobulin VH usage analysis by fluorescent in situ hybridization and flow cytometry

1992 ◽  
Vol 153 (1-2) ◽  
pp. 249-259 ◽  
Author(s):  
Kodimangalam S. Ravichandran ◽  
Anthony R. Semproni ◽  
Richard A. Goldsby ◽  
Barbara A. Osborne
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Usage Analysis

scholarly journals DETECTION AND QUANTITATION OF RED COMPLEX BACTERIA IN SUBGINGIVAL PLAQUE BY USING FLUORESCENT IN SITU HYBRIDIZATION (FISH)

2017 ◽  
Vol 5 (11) ◽  
pp. 279-289
Author(s):  
Kishore G. Bhat ◽  
Aradhana Chhatre ◽  
Vijay M. Kumbar ◽  
Manohar S. Kugaji ◽  
Sanjeevani Patil
Keyword(s):  
In Situ Hybridization ◽  
Diagnostic Tool ◽  
Fluorescent In Situ Hybridization ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Subgingival Plaque ◽  
Periodontal Pathogens ◽  
Significant Difference ◽  
Strong Linear Relationship

Motivation/Background: Red complex bacteria are proven periodontal pathogens. In dentistry, there is a need to identify and quantitate the organisms from the diseased sites quickly and reliably. Since culture requires several days, molecular methods are being used frequently to detect these bacteria.  Among them, Fluorescent in situ hybridization (FISH) is rapid, sensitive and quantitative. An attempt is made here to evaluate the applicability of this technique as a diagnostic tool in periodontology. Method: Subgingival plaque was collected from participants, fixed with paraformaldehyde and subjected to FISH. Fluorescently labeled oligonucleotide probes were used for hybridization. After the procedure, the fluorescently stained bacteria were identified and counted from the smear and quantitated using a simple grading. Results: There was a significant difference in the prevalence and numbers of red complex bacteria in healthy and diseased subjects. A strong linear relationship existed between P. gingivalis, T. forsythia and T. denticola. Conclusions: The procedure used in the study is simple, rapid and can be easily adaptable. It also has a high sensitivity and has the ability to detect a single bacterial cell. The method can be directly applied to the clinical samples and can be used as a rapid diagnostic tool in periodontics.

16S rRNA fluorescent in situ hybridization of human colon and terminal ileum biopsies: A mucus-adherent bacterial flora does not exist

2001 ◽  
Vol 120 (5) ◽  
pp. A706
Author(s):  
Laurens A. Van der Waaij ◽  
Hermie J.M. Harmsen ◽  
Mohsen Madjipour ◽  
Frans G.M. Kroese ◽  
Hendrik M. Van Dullemen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Terminal Ileum ◽  
Fluorescent In Situ Hybridization ◽  
Bacterial Flora ◽  
Human Colon

scholarly journals Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry

2007 ◽  
Vol 73 (6) ◽  
pp. 2020-2023 ◽  
Author(s):  
Jeremy Lenaerts ◽  
Hilary M. Lappin-Scott ◽  
Jonathan Porter
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Activated Sludge ◽  
River Water ◽  
Environmental Samples ◽  
Fluorescent In Situ Hybridization ◽  
Molecular Beacons ◽  
Target Cells ◽  
Linear Dna

ABSTRACT Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry.

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