Easy flat embedding of oriented samples in hydrophilic resin (LR White) under controlled atmosphere: application allowing both nucleic acid hybridizations (CARD-FISH) and ultrastructural observations.

Hydrophilic resins present the advantage of making possible both hybridization experiments involving either antibodies or oligonucleotide probes and ultrastructural observations. Whereas various embedding protocols are available, only very few concern flat-embedded preparations. In this study we describe an easy protocol for flat embedding of small-oriented biological samples in hydrophilic resins (LR White). The most important constraints are (i) to polymerize the samples under argon-saturated atmosphere (avoiding oxygen which is an inhibitor of LR White polymerization) and (ii) to use transparent flat embedding molds. Two kinds of samples were analyzed: small pieces of large tissue that need to be accurately oriented for a valuable analysis and very small organisms such as free-living nematodes, which are very hard to investigate with conventional paraffin wax embedding techniques. Semi-thin sections strongly reinforce the quality of the observations from oligonucleotidic in situ hybridization experiments by reducing the background usually encountered in oligonucleotide probe hybridization experiments from sections. Such protocols could also permit a cheap alternative to the use of laser scanning confocal microscopes for oligonucleotidic in situ hybridization as in FISH and CARD-FISH experiments from histological sections. The interest of this embedding protocol is reinforced by the fact that molecular in situ hybridization experiments and ultrastructural observations from thin sections can be carried out from a single-small individual (<1mm in length) sample.

Raman microspectrometry as a powerful tool for a quick screening of thiotrophy: an application on mangrove swamp meiofauna of Guadeloupe (F.W.I.).

The mangrove swamp environment constitutes a sulphide rich habitat harbouring some thioautotrophic organisms. The ciliate Zoothamnium niveum and the nematode Eubostrichus dianae, both known to live associated with bacterial sulphide-oxidizing ectosymbionts, were analysed as positive controls by Raman microspectrometry. The detection of the 3 Raman bands characteristic of elemental sulphur (S(8)) allows us to define a positive model of sulphide-oxidizing symbiotic invertebrates and by extrapolation, of thioautotrophic organisms. A fast screening using this tool was carried out on eukaryotic organisms such as hydrozoan, nematodes, annelids, copepods, and ciliate (Pseudovorticella sp.) and on free-living filamentous bacteria found on decomposing leaves in order to detect thioautotrophic organisms. The Raman microspectrometry permits us: (i) to reveal thioautotrophic metabolism of free-living bacteria (Beggiatoa sp.) and even for Archaea and (ii) to detect sulphide-oxidizing endosymbiotic and ectosymbiotic bacteria associated with the Bivalve Lucina pectinata and Pseudovorticella sp., respectively. Raman microspectrometry represents a fast, easy and non destructive technique which can be used on living organisms without constraints of sample size. The Raman analysis can also be completed by ultrastructural analysis (SEM, TEM) on the same sample.