The standard disease-screening technique applied to shellfish is histology (Howard and Smith, 1983; Bell and Lightner, 1988; Bucke, 1989). Although frequently insufficient for species identification of microbial pathogens (especially those with a non-specific host response), histology provides a useful record of infection foci and host response. The host-parasite interaction at the tissue and cellular level is essential for interpreting pathogenic effects. Furthermore, light microscopy is frequently required for selecting optimal tissues for ultrastructural examination.
Standard fixatives used include Davidson's fixative (Shaw and Battle, 1957; Howard and Smith, 1983; Bell and Lightner, 1988; Bucke, 1989), 10% buffered formalin and 1G4F (1 part gluteraldehyde : 4 parts formaldehyde) (C.A. Farley in Howard and Smith, 1983). Carson's fixative, which is recommeded for fixing mollusc tissues in many European laboratories, is similar to 1G4F, but uses paraformaldehyde instead of buffered formaldehyde. Some laboratories use Bouin's fixative; however, the picric acid component of the solution renders it hazardous for long-term storage, transport and disposal. Optimal fixation requires live or moribund specimens, ideally within 12 h of removal from the water, to reduce dehydration pathology changes in live specimens. Fixatives, such as buffered formaldehyde or Davidson's solution, may be used on intact soft tissues removed from the shell. Effective tissue fixation requires a fixative solution to tissue ratio of at least 10 : 1 by volume.
Shellfish larvae less than 5-10 mm in diameter are fixed whole. Bucke (1989) recommends a double-embedding technique, where preserved larvae are concentrated into a pellet, using centrifugation in molten agar. The agar is allowed to set and then embedded in paraffin. Although the agar takes up some stain (basophilic), it does not affect the staining properties of the larvae. Good results have also been achieved by placing larvae in biopsy bags (VWR Scientific, USA), which are put inside tissue cassettes (M.F. Stephenson, Gulf Fisheries Centre, Moncton, New Brunswick, Canada, 1995, personal communication) for paraffin-embedding. Bivalves less than 10-15 mm in diameter may be fixed in their shells, after severing the hinge ligament to facilitate fixative penetration of all the soft tissue. Large bivalves are removed from the shell and appropriate tissue sections taken for fixation. Non-gluteraldehyde based fixatives may be used for larger portions of tissues while glutaraldehyde-based fixatives require tissue less than 5 mm thick.
Sections containing calcareous deposits (e.g. pearls or shelled larvae) require decalcification prior to embedding and sectioning. Decalcification can be achieved using ethylenediaminetetra-acetate (EDTA) (Howard and Smith, 1983) or another chelating agent, which dissolves the calcium carbonate without adversely affecting tissue preservation. Other chelating agents include 16% 4N-formic acid, recommended by Bucke (1989) for controlled decalcification, or other commercial decalcifiers. Decalcified tissues must be rinsed thoroughly in running tapwater before dehydrating for paraffin/resin embedding.
Fixation of small crustacean tissues is via injection through the carapace to avoid tissue damage during dissection (Bell and Lightner, 1988). Anatomy diagrams in a number of different invertebrate biology references may be used for other invertebrates, e.g. Bullough (1958), Hyman (1955), Johnson (1980), Bevelander (1988) and Jangoux (1990). Following fixation, tissues are removed for embedding and sectioning.
Stains used routinely for paraffin-embedded tissue sections (Howard and Smith, 1983; Bucke, 1989) include haematoxylin and eosin, which stain nuclei red (except for the diffuse chromatin of mature ovocytes (C.A. Farley, NOAA-DNR Oxford Laboratory, Oxford, Maryland, USA, 1988 personal communication)), along with mucin, cartilage and some Gram-negative bacteria (including RLOs and Chlamydia-like organisms). Gram's methods are used to detect bacteria and various modifications are recommended, e.g. the Brown and Brenn method, which stains Gram-positive organisms blue (e.g. Nocardia, Gaffkemia, Micrococcus) and Gram-negative organisms red, while tissues appear yellow. The Giemsa method stains Rickettsia and Chlamydia parasites blue-black, nuclei are blue and other structures are pink or light blue. Grocott's modified Gomori chromic acid, methenamine silver-nitrate stain (Grocott's methenamine silver (GMS)), is recommended for fungi (Bucke, 1989), which stain black (as do Gram-positive bacteria, such as Nocardia sp. and Aerococcus sp.). Mucin stains brown and background tissues stain light green. Farley's Feulgen picromethyl blue stain (Howard and Smith, 1983) is recommended to differentiate DNA from RNA and is useful for viral-like intracellular inclusions. Deoxyribonucleic acid stains red, RNA yellow, connective tissue blue and muscle and cytoplasm yellow/green. Double-stranded DNA and single-stranded nucleic acids (including RNA) are differentiated histochemically, using acridine orange fluorescence (Gale et al., 1981). Malt PAS (MPAS) is used for PASpositive reactions with cellulose and starch, notably in fungal tissue infections.
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