Methods 



Microstructural increments can be studied in thin sections of shell material, 

 in acetate peel repUcas of acid-etched shell sections, or under the scanning 

 electron microscope (fractured or polished and etched shell sections). 



Acetate peels are the easiest and most rapid method of preparation for 

 examination of most molluscan shells. The basic method of preparation, as 

 outlined by Rhoads and Pannella (32), is as follows: 



Shells are embedded in a block of epoxy resin (e.g., Epon 815 resin with 

 DTA liardener, 10:1 ratio, under vacuum; Miller-Stephenson Chemical 

 Company, Danbury, Connecticut) to avoid shell fracture during sectioning. The 

 plane of the cross-section passes from the umbo to the shell edge along the axis 

 of maximum growth (30, 32). This cut is oriented so that growth increments 

 intersect the plane of the section at right angles. The cut shell surface is 

 polished sequentially with 350, 600, and, finally, 2600 or 3000 grade 

 carborundum grits. The polished surface is then etched with 0.1 N HCl for 

 periods varying from a few seconds to a few minutes. Optimal etching time is 

 related to shell structure, mineralogy, organic content, and state of 

 preservation. It is recommended that a series of test etching times be carried 

 out to determine optimum etching periods for a particular set of specimens. 



Etched shell surfaces are flooded wdth acetone, and a piece of sheet acetate 

 is applied to the etched shell surface and weighted to avoid bubble formation. 

 After the acetone (solvent) has evaporated (approximately 30 minutes), the 

 acetate is removed from the shell and examined under the microscope (or used 

 as a negative by placing directly in a photographic enlarger and printing). Tliis 

 technique yields excellent results for most species. 



Thin-sections are necessary for the examination of growth increments which 

 are not structurally discontinuous, but instead recognizable only by dark and 

 light color bands (32). For example, the growth increment boundaries in the 

 deep-water species, Nuada cancellata and Calyptogena ponderosa, are 

 indistinct and recognizable only by color variations of the bands, each band 

 consisting of one dark and one light layer. The initial procedure for making 

 thin-sections is the same as that for preparation of acetate peels, however, after 

 the cut shell surface has been polished, it is glued to a glass slide using epoxy 

 resin. The majority of the embedded shell and remaining embedding material is 

 cut away using a diamond rock cutting saw, and the new exposed surface is 

 polished sequentially until a 0.03 mm thick section of material is left on the 

 sHde. A cover slip is glued onto the newly poHshed surface using epoxy resin, 

 and the material examined using optical or polarizing microscopy. Thin 

 sectioning of shells is difficult, because shells tend to fracture when sectioned 



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