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HISTOLOGICAL PREPARATION OF INVERTEBRATES FOR EVALUATING CONTAMINANT EFFECTS
Peters, E., K. Price, AND D B. Horowitz. HISTOLOGICAL PREPARATION OF INVERTEBRATES FOR EVALUATING CONTAMINANT EFFECTS. Chapter 36, GK Ostrander (ed.), Techniques in Aquatic Toxicology, Volume II. CRC Press - Taylor & Francis Group, LLC, Boca Raton, FL, , 653-686, (2005).
Although many studies in toxicologic pathology evaluate the effects of toxicants on fishes because of their similarities with other vertebrates, invertebrates can also provide insights into toxicant impacts on ecosystems. Invertebrates not only serve as food resources (e.g., worms, clams, shrimp, insects), but as agents of physical habitat change (e.g., reef-building corals, bioeroding sponges, bioturbating worms), among other roles. Adverse effects on individuals and populations of any species due to toxicant exposures from water, sediment, or food can ultimately alter community relationships and have direct and indirect impacts on members of the community. Many invertebrates are sedentary or have a limited range of mobility compared to fishes. Because of this, their health may reflect local environmental conditions better than fishes. Therefore, invertebrates can be used to study chronic or acute exposures to single toxicants or mixtures in the field and laboratory. However, many such studies of invertebrate species limit their observations to gross descriptions, percent of organisms killed by the toxicant, or reductions in growth and reproduction. Knowing that an animal died or did not reproduce, however, may not be as important as knowing why. Toxicant effects can be measured using many techniques and tools, but the only techniques that can provide visual images of the internal condition of the cells and tissues of an organism are observations made using light and electron microscopy. This field is known as histology. Structure and composition of cells and tissues reflect their function and overall functioning of the organism. Histotechniques provide data that can be used in conjunction with procedures from other fields (e.g., physiology, biochemistry, molecular biology, ecology) to improve our understanding of invertebrate susceptibilities to contaminants, mechanisms of toxicant damage to target cells and organs, and impact of the exposure on the individual, population, and community. This chapter will discuss the preparation of invertebrate tissues for light microscopical examination using paraffin embedding, a common procedure in many hospital and veterinary medical diagnostic operations. The resulting product is a section of tissue typically 2–10 ?m thick mounted on a glass microscope slide (“histoslide”). Dyes or other compounds can be used to enhance particular features of tissues for study with bright-field illumination or epifluorescence. Although basic steps for tissue fixation, dehydration, clearing, embedding, sectioning, and staining have remained similar since the late 1800s, recent developments in equipment, techniques, and the ability to identify specific molecules in tissues could require different approaches to improve the microscopic appearance of tissues or to address specific research questions. Three kinds of data can be generated from histoslides to test hypotheses: descriptive, qualitative, and quantitative data. Study objectives must be established prior to collecting and processing organisms to identify the most appropriate methodology for producing the required information (i.e., the correct type, quantity, and quality of histological data). The hypothesis(es) being tested and the endpoints (data) that will be collected using microscopic examinations of the stained tissue sections should be framed. Qualitative (ranked) data can be compared using non-parametric statistics and quantitative data can be compared using parametric statistics. Adequate numbers of control (unexposed) animals need to be included in every study for comparison with exposed animals. The number of replicates needed from exposed and “pristine” sites, or from laboratory exposures and controls, should be statistically determined to ensure valid data. In the case of endangered or threatened species, such as stony corals, a minimum of three samples from different colonies from each exposure concentration should be examined to provide an indication of individual variability and normal tissue conditions within that field site or experiment. You should plan to collect the invertebrates from the cleanest water first, then sample from the least to most contaminated, rinsing equipment between samples. Also consider limitations on resources versus data needs, i.e., how many histoslides of which portions of tissue will you need to examine to test the hypothesis(es), versus what will you be able to examine based on time, expertise, or funding constraints. Since the ability to obtain the data is dependent on the quality of the histoslide produced, this procedure focuses on two steps: Fixation to preserve the tissues without postmortem autolysis, and staining to enable the observer to see the structure and composition of the cells and tissues so that comparisons can be made with established criteria for cell damage, or with control specimens. Interpreting structure and composition seen in the tissue section under the microscope to derive these data requires special training, which cannot be covered in this chapter.