Flow sorting of microorganisms for molecular analysis

Not only classical cultivation-based methods but also the new molecular approaches may result in incomplete and selective information on the natural diversity of microbial communities. Flow sorting of microorganisms from environmental samples allows the deliberate selection of cell populations of interest from highly diverse systems for molecular analysis. Several cellular parameters that can be measured by flow cytometry are useful as sort criteria. Here, we report sorting of bacteria from activated sludge, lake water, and lake sediment according to differences in light scattering, DNA content, and/or affiliation to certain phylogenetic groups as assessed by fluorescein-labeled, rRNA-targeted oligonucleotide probes. Microscopy of the sorted cells showed that populations of originally low abundance could be strongly enriched by flow sorting (up to 280-fold), depending on the original abundance of the cells of interest and the type of sample sorted. The purity of the cells of interest could be further increased by repeated sorting, but this increase was limited by cell aggregation in the case of activated-sludge samples. It was possible to amplify almost full-length 16S ribosomal DNA (rDNA) fragments from sorted microbial cells by PCR, even after fixation with paraformaldehyde and in situ hybridization. Dot blot hybridization and sequencing demonstrated that most of the amplified rDNA originated from those cells that had been selected for by flow sorting. Comparative analysis of 16S rDNA sequences revealed previously unknown species of magnetotactic or activated-sludge bacteria.

[1]  N. Pace,et al.  Microbial ecology and evolution: a ribosomal RNA approach. , 1986, Annual review of microbiology.

[2]  M. Urdea,et al.  A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes. , 1988, Nucleic acids research.

[3]  K. Schleifer,et al.  Phylogenetic Diversity and Identification of Nonculturable Magnetotactic Bacteria , 1992 .

[4]  K. Schleifer,et al.  Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge , 1994, Applied and environmental microbiology.

[5]  D. Deere,et al.  Fluorescent probes and flow cytometry: new insights into environmental bacteriology. , 1996, Cytometry.

[6]  W. Nader,et al.  Detection and analysis of two serotypes of ammonia-oxidizing bacteria in sewage plants by flow cytometry , 1990, Applied and environmental microbiology.

[7]  H. Noller,et al.  Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. , 1981, Journal of molecular biology.

[8]  M. Moran,et al.  In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities , 1995, Applied and environmental microbiology.

[9]  R. Huber,et al.  Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis , 1995, Nature.

[10]  A. Uitterlinden,et al.  Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA , 1993, Applied and environmental microbiology.

[11]  R. Amann,et al.  Flow cytometric analysis of activated sludge with rRNA-targeted probes , 1995, Applied and environmental microbiology.

[12]  D. Schüler,et al.  Phylogenetic Analysis of Uncultured Magnetotactic Bacteria from the Alpha-Subclass of Proteobacteria , 1995 .

[13]  K. Schleifer,et al.  Identification and in situ Detection of Gram-negative Filamentous Bacteria in Activated Sludge , 1994 .

[14]  V. Skerman.  A new type of micromanipulator and microforge. , 1968, Journal of general microbiology.

[15]  Y. Zo,et al.  Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism , 1996, Applied and environmental microbiology.

[16]  C. Edwards,et al.  Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction. , 1995, FEMS microbiology letters.

[17]  D Marie,et al.  Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry , 1995, Applied and environmental microbiology.

[18]  D A Stahl,et al.  Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology , 1990, Journal of bacteriology.

[19]  R Amann,et al.  Phylogenetic analysis and in situ identification of bacteria in activated sludge , 1997, Applied and environmental microbiology.

[20]  H B Steen,et al.  Noise, sensitivity, and resolution of flow cytometers. , 1992, Cytometry.

[21]  P Simonet,et al.  Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction , 1992, Applied and environmental microbiology.

[22]  B. Robertson,et al.  Characterizing aquatic bacteria according to population, cell size, and apparent DNA content by flow cytometry. , 1989, Cytometry.

[23]  M. F. Sialer,et al.  Formalin-induced infidelity in PCR-amplified DNA fragments. , 1994, Molecular and cellular probes.

[24]  E. Delong,et al.  Application of rRNA-based probes for observing marine nanoplanktonic protists , 1993, Applied and environmental microbiology.

[25]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[26]  K. Schleifer,et al.  Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure , 1993, Applied and environmental microbiology.

[27]  O. C. Blair,et al.  Practical Flow Cytometry , 1985, The Yale Journal of Biology and Medicine.

[28]  R. Amann,et al.  Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations , 1990, Applied and environmental microbiology.

[29]  D. Kell,et al.  Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. , 1996, Microbiological reviews.

[30]  J H Jett,et al.  Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones. , 1996, Nucleic acids research.

[31]  K. Schleifer,et al.  Dominating Role of an Unusual Magnetotactic Bacterium in the Microaerobic Zone of a Freshwater Sediment , 1993, Applied and environmental microbiology.

[32]  R. Amann,et al.  Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. , 1993, Cytometry.

[33]  K. Schleifer,et al.  Phylogenetic Oligodeoxynucleotide Probes for the Major Subclasses of Proteobacteria: Problems and Solutions , 1992 .

[34]  N. Pace,et al.  Differential amplification of rRNA genes by polymerase chain reaction , 1992, Applied and environmental microbiology.

[35]  Frank Oliver Glöckner,et al.  An in situ hybridization protocol for detection and identification of planktonic bacteria , 1996 .

[36]  K. Schleifer,et al.  In situ visualization of high genetic diversity in a natural microbial community , 1996, Journal of bacteriology.

[37]  R. Amann,et al.  Combination of rRNA-Targeted Hybridization Probes and Immuno-Probes for the Identification of Bacter , 1996 .

[38]  E. Chen,et al.  Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. , 1985, DNA.

[39]  F. Jüttner,et al.  Microflora of a subalpine lake: bacterial populations, size and DNA distributions, and their dependence on phosphate , 1996 .

[40]  R. Amann,et al.  Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification , 1997, Applied and environmental microbiology.

[41]  H. Steen,et al.  Light scattering measurement in an arc lamp-based flow cytometer. , 1990, Cytometry.