Titre du document / Document title

An improved method for single cell isolation of prokaryotes from meso-, thermo- and hyperthermophilic environments using micromanipulation

Auteur(s) / Author(s)

ISHØY Thomas ⁽¹⁾ ; KVIST Thomas ⁽¹⁾ ; WESTERMANN Peter ⁽¹⁾ ; AHRING Birgitte K. ⁽¹⁾ ;

Affiliation(s) du ou des auteurs / Author(s) Affiliation(s)

⁽¹⁾ Environmental Microbiology and Biotechnology Research Group (EMB), BioCentrum-DTU, Technical University of Denmark, Building 227, 2800, DANEMARK

Résumé / Abstract

This study presents an improved system that enables isolation of single viable prokaryotic cells from a mixture of cells. The system is based on an inverted microscope, a microinjector and a micromanipulator. The isolatedcell is captured in a microcapillary from a volume of 400 μl and transferred to an appropriate growth medium. Validation of the system was performed using two fluorescent strains: Pseudomonas putida expressing red fluorescent protein (DsRed), and Escherichia coli expressing green fluorescent protein (GFP). A mixture (100:1) of the constructed fluorescent strains was subjected to isolation experiments and nine out of ten individually isolated cells yielded axenic cultures of E. coli. Upon construction and validation, the system was used to isolate and subsequently cultivate axenic cultures of the thermophilic Archaeon Metallosphaera sedula and the hyperthermophilic Archaeon Sulfolobus solfataricus from enriched hot spring samples. The high efficiency of single-cell isolation and cultivation demonstrated over a range of temperatures-90% (30°C), 85% (70°C) and 95% (80°C)-from different environments is probably due to the elimination of osmotic stress and limitation of temperature fluctuations during the isolation process, as a result of the large sample volume from which the cells are isolated.

Revue / Journal Title

Applied microbiology and biotechnology    ISSN  0175-7598   CODEN AMBIDG 

Source / Source

2006, vol. 69, n^o5, pp. 510-514 [5 page(s) (article)] (22 ref.)

Langue / Language

Anglais

Editeur / Publisher

Springer, Heidelberg, ALLEMAGNE  (1984) (Revue)

Mots-clés anglais / English Keywords

Thermophily

;

Isolated cell

;

Bacteria

;

Procaryote

;

Micromanipulation

;

Hyperthermophily

;

Isolation

;

Method

;

Mots-clés français / French Keywords

Thermophilie

;

Cellule isolée

;

Bactérie

;

Procaryote

;

Micromanipulation

;

Hyperthermophilie

;

Isolement

;

Méthode

;

Mots-clés espagnols / Spanish Keywords

Termofilia

;

Célula aislada

;

Bacteria

;

Procariota

;

Micromanipulación

;

Hipertermofilia

;

Aislamiento

;

Método

;

Localisation / Location

INIST-CNRS, Cote INIST : 16771, 35400013474698.0050