An Improvement in the Protargol Technique of Ng and Nelsen

Abstract

A simple modification of the Protargol staining technique of Ng and Nelsen gives significantly improved results. This consists of adding anhydrous Na2CO3 to give a concentration of 4% (w/v) to the developer solution. The modification inhibits mitochondrial staining and improves the differentiation between the cortical microtubular structures and the background cytoplasm. The Protargol staining technique described by Ng & Nelsen (1977) produces results of sufficient reliability and sensitivity to be useful in the study of cortical morphogenesis of members of the Tetrahymena pyriformis species group (Nanney & McCoy, 1976); several important papers using this technique have appeared (e.g., Ng & Frankel, 1977; Ng, 1978, 1979a,b; JerkaDziadosz & Frankel, 1979). Staining tests indicate that this technique produces the best results when the Protargol manufactured by E. Merck is used. However, most lots of Merck Protargol will stain mitochondria in addition to microtubular structures and nuclei. Although the simultaneous staining of mitochondria and cortical microtubules in the same cell has been exploited in studies of cortical patterning of mitochondria in Tetrahymena thermophila (Aufderheide, 1979, 1980), most researchers would prefer to eliminate the mitochondrial staining and stain only the microtubular structures. A simple modification of the Protargol technique of Ng & Nelsen, which allows the researcher to encourage or suppress the mitochondrial staining, is reported here. This modification also significantly improves the quality and repeatability of the staining of the cortical microtubular structures, and already has been used in several studies (Aufderheide, 1979, 1980; Nelsen & Frankel, 1979). MATERIALS AND METHODS Cells of Tetrahymena thermophila (stock B), Tetrahymena pyriformis (stock GL-C), and Tetrahymena paravorax (stock RP) were grown axenically in 1% proteose-peptone, 0.1% yeast-extract medium (PPY). Cultures were maintained at 30?C. For staining, cells were harvested from cultures 1-12 days post-inoculation. The Protargol technique described by Ng & Nelsen (1977) was followed 1 The research for this paper was supported by Texas A&M University Minigrant No. 15707 and in part by NIH Grant HD 08485 to J. Frankel. The author acknowledges the generous help of Stephen Ng in sharing his staining test results and his critical reading of the manuscript. TRANS. AM. MICROSC. SOC., 101(1): 100-104. 1982. (c) Copyright, 1982, by the American Microscopical Society, Inc. This content downloaded from 207.46.13.115 on Sat, 08 Oct 2016 04:16:41 UTC All use subject to http://about.jstor.org/terms VOL. 101, NO. 1, JANUARY 1982 in detail, except for the specific modifications described below. Protargol (Silver, Protein, Catalog no. 7447) was purchased from E. Merck, Danmstadt, BRD (U.S. distributor: Harleco, Attn: Bob Kolacki, 480 Democrat Rd., Gibbstown, New Jersey 08027). The following lot numbers have been tested: 6950443, 8580399, and 9638484. RESULTS AND DISCUSSION