Staining and Mounting Helminths

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Abstract
The following procedure is recommended: Fix ces-todes and trematodes (while held flat between glass slides) 0.5–2.0 hr. in the following mixture: formalin, 15; acetic acid (gl.), 5; glycerol, 10; 95% ethyl alcohol, 24; distilled H2O, 46; all proportions by volume. After freeing them from the slides, wash thoroughly in running water and stain immediately thereafter. Stock staining solution: ferric ammonium alum (violet cryst.), 2 g.; distilled H2O (cold) 100 ml.; after solution, add 2 ml. concentrated H2SO4, bring to a boil; add 1 g. coelestin blue B (Nat. Aniline), boil 3–5 min.; cool and add 10 ml. absolute methyl alcohol and 10 ml. glycerol. Dilute 1 vol. with 3 vol. distilled H20 for use. Stain 5–30 min., depending on size of specimens. Wash with 2 changes 0.5 hr. each of distilled H2O, then 50% isopropyl alcohol 12–16 hr., 50% isopropyl alcohol 2 hr., followed by graded isopropyl alcohol for dehydration. Ether: ethyl alcohol (equal parts), 1 hr., is followed by embedding in celloidin in a sheet just thick enough to cover the specimens. Trim embedded specimens and dehydrate with isopropyl alcohol, 80%, 90% and absolute. Clear in beechwood creosote. Mount in balsam with cover glasses that overlap the edges of the celloidin 1–2 mm. While drying at 37°C, refill edges of mount with fresh balsam as needed. When dry, remove excess balsam and ring the edges with ordinary gloss enamel paint.

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