A Correlative Light-Electron Microscopy (CLEM) Protocol for the Identification of Bacteria in Animal Tissue, Exemplified by Methanotrophic Symbionts of Deep-Sea Mussels

2015 ◽  
pp. 163-174 ◽  
Author(s):  
Sven R. Laming ◽  
Sébastien Duperron
Keyword(s):  
Electron Microscopy ◽  
Deep Sea ◽  
Animal Tissue ◽  
Identification Of Bacteria

Application of ruthenium red/osmium tetroxide to bacteria, plant and animal tissue

1986 ◽  
Vol 44 ◽  
pp. 54-55
Author(s):  
R. L. Grayson ◽  
N. A. Rechcigl
Keyword(s):  
Electron Microscopy ◽  
Electron Density ◽  
Osmium Tetroxide ◽  
Biological Materials ◽  
Ruthenium Red ◽  
Animal Tissue

Ruthenium red (RR), an inorganic dye was found to be useful in electron microscopy where it can combine with osmium tetroxide (OsO4) to form a complex with attraction toward anionic substances. Although Martinez-Palomo et al. (1969) were one of the first investigators to use RR together with OsO4, our computor search has shown few applications of this combination in the intervening years. The purpose of this paper is to report the results of our investigations utilizing the RR/OsO4 combination to add electron density to various biological materials. The possible mechanisms by which this may come about has been well reviewed by previous investigators (1,3a,3b,4).

scholarly journals Localization of ‘Candidatus Liberibacter solanacearum’ and Evidence for Surface Appendages in the Potato Psyllid Vector

2016 ◽  
Vol 106 (2) ◽  
pp. 142-154 ◽  
Author(s):  
J. M. Cicero ◽  
T. W. Fisher ◽  
J. K. Brown
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Fluorescence Labeling ◽  
Potato Psyllid ◽  
Bactericera Cockerelli ◽  
Visual Identification ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Identification Of Bacteria

The potato psyllid Bactericera cockerelli is implicated as the vector of the causal agent of zebra chip of potato and vein-greening of tomato diseases. Until now, visual identification of bacteria in the genus ‘Candidatus Liberibacter’ has relied on direct imaging by light and electron microscopy without labeling, or with whole-organ fluorescence labeling only. In this study, aldehyde fixative followed by a coagulant fixative, was used to process adult psyllids for transmission electron microscopy (TEM) colloidal gold in situ hybridization experiments. Results indicated that ‘Ca. Liberibacter solanacearum’ (CLso)-specific DNA probes annealed to a bacterium that formed extensive, monocultural biofilms on gut, salivary gland, and oral region tissues, confirming that it is one morphotype of potentially others, that is rod-shaped, approximately 2.5 µm in diameter and of variable length, and has a rough, granular cytosol. In addition, CLso, prepared from shredded midguts, and negatively stained for TEM, possessed pili- and flagella-like surface appendages. Genes implicating coding capacity for both types of surface structures are encoded in the CLso genome sequence. Neither type was seen for CLso associated with biofilms within or on digestive organs, suggesting that their production is stimulated only in certain environments, putatively, in the gut during adhesion leading to multiplication, and in hemolymph to afford systemic invasion.

Predation on copepods by an Alaskan cladorhizid sponge

2007 ◽  
Vol 87 (6) ◽  
pp. 1721-1726 ◽  
Author(s):  
Les Watling
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Lipid Content ◽  
Deep Sea ◽  
Prey Capture ◽  
Primary Process ◽  
High Lipid ◽  
Very High ◽  
Prey Items ◽  
Scanning Electron

Deep water cladorhizids are now generally thought to be carnivorous. While the methods of prey capture have been established for species of Asbestopluma, and perhaps Chondrocladia, information is sparse for species of Cladorhiza. The external morphology of the deep-sea Alaskan species, Cladorhiza corona, was examined immediately after collection, and then again later using scanning electron microscopy (SEM). All sponges examined had captured, and in most cases, fully encased, prey items which for the most part consisted of planktonic copepods. The hypothesis of this paper is that the primary process of prey capture is by means of a sticky substance and that prey stick to the sponge on contact. The abundant cladorhizid anisochelae do not seem to be arranged in a manner such that they can be used for prey capture as has been seen in Asbestopluma. Digestion of the prey occurs in a temporary cavity created by migrating sponge cells. Within this cavity lipid from the copepod is liberated, then absorbed and transported to the central part of the sponge. It is not known how much of the remaining tissue of the copepod is digested. Copepods have very high lipid content so their capture would provide the sponge with abundant energy from each prey item. The sponges seem to have their highest density in areas where bottom flow and migrating copepod numbers are both high.

Sponge fauna associated with a Mediterranean deep-sea coral bank

2005 ◽  
Vol 85 (6) ◽  
pp. 1341-1352 ◽  
Author(s):  
Caterina Longo ◽  
Francesco Mastrototaro ◽  
Giuseppe Corriero
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Mediterranean Sea ◽  
Endemic Species ◽  
Deep Sea ◽  
Southern Italy ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
First Time ◽  
Scanning Electron

Thirty species of sponges (29 Demospongiae, 1 Hexactinellida) have been recorded in association with a white coral bank situated off Cape S. Maria di Leuca (southern Italy) at depths ranging from 430 to 1160 metres. Notwithstanding the occurrence of clearly eurybathic species, two depth-dependent sponge groups can be identified along the bathymetric gradient. Two species, Geodia nodastrella and Plocamiopsis signata, are reported for the first time from the Mediterranean Sea. The sponge assemblage shows a higher affinity with the fauna from the Boreal region, with very low number of Mediterranean endemic species. Systematic notes concerning the poorly known and intriguing species, studied using scanning electron microscopy analysis, are reported.

scholarly journals Techniques in Electron Microscopy of Animal Tissue

2013 ◽  
Vol 51 (1) ◽  
pp. 28-41 ◽  
Author(s):  
N. F. Cheville ◽  
J. Stasko
Keyword(s):  
Electron Microscopy ◽  
Animal Tissue

scholarly journals Direct Image-Based Correlative Microscopy Technique for Coupling Identification and Structural Investigation of Bacterial Symbionts Associated with Metazoans

2011 ◽  
Vol 77 (12) ◽  
pp. 4172-4179 ◽  
Author(s):  
Sébastien Halary ◽  
Sébastien Duperron ◽  
Thomas Boudier
Keyword(s):  
Electron Microscopy ◽  
Deep Sea ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Type I ◽  
Ultrastructural Investigation ◽  
Microscopy Technique ◽  
Bacterial Morphology ◽  
Single Host

ABSTRACTCoupling prokaryote identification with ultrastructural investigation of bacterial communities has proven difficult in environmental samples. Prokaryotes can be identified by using specific probes and fluorescencein situhybridization (FISH), but resolution achieved by light microscopes does not allow ultrastructural investigation. In the case of symbioses involving bacteria associated with metazoan tissues, FISH-based studies often indicate the co-occurrence of several bacterial types within a single host species. The ultrastructure is then relevant to address host and bacterial morphology and the intra- or extracellular localization of symbionts. A simple protocol for correlative light and electron microscopy (CLEM) is presented here which allows FISH-based identification of specific 16S rRNA phylotypes and transmission electron microscopy to be performed on a same sample. Image analysis tools are provided to superimpose images obtained and generate overlays. This procedure has been applied to two symbiont-bearing metazoans, namely, aphids and deep-sea mussels. The FISH protocol was modified to take into account constraints associated with the use of electron microscopy grids, and intense and specific signals were obtained. FISH signals were successfully overlaid with bacterial morphotypes in aphids. We thus used the method to address the question of symbiont morphology and localization in a deep-sea mussel. Signals from a type I methanotroph-related phylotype were associated with morphotypes displaying the stacked internal membranes typical for this group and three-dimensional electron tomography was performed, confirming for the first time the correspondence between morphology and phylotype. CLEM is thus feasible and reliable and could emerge as a potent tool for the study of prokaryotic communities.

Setal morphology in cirripedes: a useful tool in phylogenetic studies?

2011 ◽  
Vol 92 (2) ◽  
pp. 305-325 ◽  
Author(s):  
L.J. Walley
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Shell Structure ◽  
Deep Sea ◽  
Littoral Zone ◽  
Dna Analysis ◽  
Soft Body ◽  
Barnacle Species ◽  
Phylogenetic Studies ◽  
Scanning Electron

The morphology of the setae, on the mouthparts and the cirri of 33 barnacle species, was investigated by scanning electron microscopy. The species examined were selected from a range of pedunculate and balanomorph families living in a variety of habitats from the littoral zone to deep-sea vents. It was found that the setae could be divided into five groups: simple, denticulate, setulate, plumo-denticulate and aesthetascs. The categories were subdivided resulting in 43 setal types being recognized. Balanomorph cirripedes were found to have a greater variety of setae than pedunculates. Setulate setae on the cirri, and specialized setae on the mandibular palps and the first maxillae tended to be associated with the development of the balanomorph form. Setal type was correlated with taxonomy, whether based on shell structure and soft body morphology or DNA analysis, but with some anomalies which deserve further investigation. The problems of reconciling the proposed cirripede setal categories with those described in the Decapoda are discussed.

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