Addition of 0.05(y0 Os04 to a conventional glutaraldehyde fixative for the first 10 min of fixation was found to improve greatly the preservation of ultrastructure in the eggs of Urechis caupo. Several workers have since confirmed this result in other marine invertebrate tissues. Specific protocols and techniques are given. We believe that the OsO4 rapidly renders the plasma membranes of cells freely permeable to glutaraldehyde, allowing faster penetration of this fixative. This method should be applicable to a wide variety of tissues that are difficult to fix.The ultrastructure of marine invertebrate tissues, especially that of eggs, has proved difficult to preserve well (Meredith Gould-Somero & Linda Holland, personal communication, and 1975 ;Harris, 1978; Parris M. Kidd, personal communication). T o study the early development of Urechis caupo (an echiuroid worm), we tried a variety of fixation methods. Two types of methods proved to be significantly better than the others.Our first success was with a method we call a 'sequential mixed fixative'. We are using the term 'mixed fixative' in the same sense as Hirsch & Fedorko (1968) to signify a freshly prepared mixture of glutaraldehyde and 0~0 4 .The eggs were first fixed for about 5 min in 1% a s 0 4 in a mixture of NaCl and sea water (1 part 0.45 M NaCl: 1 part sea water); they were then fixed for 1-3 h in 2.5% glutaraldehyde in a NaC1-sea water solution, with or without 0.5% 0~0 4 . Post-fixation was in a fresh change of the same solution used for initial fixation. The first sequential mixed fixative formula that we tried had previously been used by Diane Gowdy (personal communication) on sea urchin embryos. A similar procedure has also been used to fix the metachronal wave of cilia (Warner & Satir, 1974).Our second method, which we call the 'low osmium mixed pre-fixative technique', gave much better preservation of ultrastructure. This method consists of a brief (5-10 min) pre-fixation in a glutaraldehyde fixative to which a small amount of Os04 (0.05%) has been added immediately before use. Fixation is then continued in the same fixative without the added oso4 for 1 h. After a brief buffer rinse, a conventional Os04 post-fixation follows.Our best results so far have been achieved with a main fixative composed of 476 glutaraldehyde, 0.2 M Na cacodylate, 0.1 M NaCl (or 11 mM CaC12,56 mM MgClz), 0.35 M sucrose, pH 7.2 (or 7.8). Post-fixation was in 1 yo Os04, 0 . 3~ NaC1,0.2~ Na cacodylate, pH 7.2. The pre-fixative was prepared by adding a small measured volume of the post-fixative to a measured aliquot of the main fixative (usually 0.5 ml added to 10.0 ml of main fixative). The pre-fixative must be mixed within 5 min of the time it is used. The buffer rinse after the main fixative consisted of two changes (5-10 min each) of 0.3 NaCl in 0.2 M Na cacodylate, pH 7.2.All of the fixation methods we tried preserved the vitelline coat of Urechis eggs well, but only the sequential mixed fixative methods and the low osmium mixed pre-fixative methods preserved the cyto...
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