Lake Pavin History, geology, biogeochemistry, and sedimentology of a deep meromictic maar lake

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Step 1: CARD-FISH

FISH is a printing method targeting classically the cel-

lular ribosomal RNA (rRNA) by hybridization of a probe

complementary to the rRNA sequence of a targeted cell.

The probe is labeled with a fluorochrome allowing the

observation by epifluorescence microscopy. CARD-

FISH combines CARD (catalysed reported deposition)

of fluorescently labeled tyramides with single-cell iden-

tification by FISH. The hybridization involves a single

oligonucleotide that is covalently crosslinked to a horse-

radish peroxidase (HRP) label. Amplification of the sig-

nal relative to that achieved with probes that are labeled

with a single fluorochrome is based on the radicalization

of multiple tyramide molecules by a single horseradish

peroxidase (Amann and Fuchs 2008 ).

Step 2: Magnetic capture

To induce the cellular capture after cell hybridization,

monoclonal mouse anti-fluorochrome-antibody labeled to

paramagnetic beads are added. A complex labeled cell-

paramagnetic bead is created and those complexes are

“captured” by the application of a magnetic field (with a

magnet) allowing to enrich a cellular fraction of targeted

cells in environmental samples.

Box 16.3 (continued)

Step 3: Observation of “captured”-cells by transmission

electron microscopy (TEM)

TEM is a useful method to appreciate the cell ultrastruc-

ture and the viral infection of cells. A key problem to

determine the cellular infection of a targeted microbial

community (e.g., Euryarchaeota) is that all the prokary-

otic community is observed in an environmental sample

by TEM without any information on their identity. We

managed to perform magnetoFISH (using oligonucleotide

probes designed for targeted group such as Euryarchaeota)

prior to TEM observations. First results show that both

methods are compatible.

Further work will be done to determine the frequency

of visible infected cells of different groups of methano-

gens but also to investigate the morphological charac-

teristics (by TEM and confocal laser scanning

microscopy (CLSM)) of targeted cells and the genetic

potential (by metagenomic) of some uncultivated

groups of microorganisms (e.g., MBGD) encountered

in Lake Pavin.

A.-C. Lehours et al.