Improvement of silver impregnation technique (Protargol) to obtain morphological features of protists ciliates, flagellates and opalinates

Abstract

The research on ciliates, flagelates and opalinates have been widespread by the utilization of techniques employing silver impregnation (Protargol), modified by several authors. However, these are time consuming and its results are variable. The present work is a variant of the technique described by Tuffrau (1964Tuffrau ( , 1967 showing some adaptations made in our laboratory. The organisms can be preserved by different fixatives (alcoholic Bouin, Stieve's fluid, 2.5% glutaraldehyde and others) and then rinsed in destilled water followed by a fast clarification by 3% sodium hypochloride. If the organism is very sensitive to hypochloride, 4% sodium lauryl sulfate may be used and then washed 3 times in distilled water. The protista can be adhered to the glass slides with Mayer's glycerinated-albumin (1 glycerin vol. to 1 or 2 albumin vol.), diluted in water at a proportion of 1:10 Cv/v., or with 1% polylysine followed by fast washes with distilled water. After the slide preparation, they were covered with a layer of 0,8% Silver proteinate. Right after that, the slide has to be placed in a glass tray lined with moist tissue and covered to prevent the proteinate to dry. The tray was placed in a incubator at 40º-50ºC for 30 minutes. The slides are rinsed for 1 minute. with warm (35ºC) distilled water. The development of the material should be done with 0.4% hydroquinone with a maximum incubation time of 1 minute. It should be developed gradually, controlling the silver impregnation intensity by observation under optical microscope. Next, rinse in distilled water for 1 minute, and then, fix in 2,5% Sodium thiosulfate. Rinse the slide for two minutes before dehydrating it in an alcoholic serial 50-100º. Finally rinse the slides in xylene. Mount the slides with Entellan MerckTM or Canada balsam.Key words: impregnation of protists, protargol, technique improvement. RESUMOAperfeiçoamento da técnica de impregnação pela prata (protargol) para obtenção de caracteres morfológicos de protistas ciliados, flagelados e opalinatas A pesquisa sobre ciliados, flagelados e opalinatas vem sendo ampliada com utilização da técnica de impregnação pela prata (protargol), modificada por vários autores. Mas, geralmente, estas demoram muito para se desenvolver e os resultados são variados. O presente trabalho é uma variante da técnica descrita por Tuffrau (1964Tuffrau ( , 1967, mostrando algumas modificações feitas no laboratório. Os organismos podem ser preservados por diferentes fixadores (Bouin alcóolico, solução de Stieves, glutaraldeído a 2,5% e outros), lavados com água destilada e em seguida clarificados com hipoclorito de sódio a 3%. Se o organismo é muito sensível ao hipoclorito, podemos fazer uso do lauril sulfato de sódio a 4% e 3 lavagens com água destilada. Os protistas são aderidos à lâmina com albuminaglicerinada de Mayer (1 vol. glicerina para 1 ou 2 vol. albumina), diluída em água na proporção de 1:10, ou com polilisina a 1%. Depois da preparação e secagem das lâminas, estas são cobertas por uma fina camada de prote...

Spatial and Temporal Distribution of Protozoa at Cueva de Los Riscos, Querétaro, México

“…To identify the protozoa and for a photomicrographic record, all the cultures were periodically examined by using phase-contrast, differential-interference-contrast and bright-field microscopes (Nikon Labophot-2 with Nikon Digital Sight DS-2Mv and Nikon FX-35DX incorporated camera; Zeiss Axioskop 2 plus with Zeiss AxioCam MRC system). Diagnostic characters were studied with staining and impregnation techniques such as Harris hematoxylin, Klein, protargol, and butanolnigrosine (Borror, 1969;Kudo, 1971, p. 863;Lee et al, 1985;Silva-Neto, 2000).…”

An inquiry into the morphology of Ciliate Protozoa using an engineering design approach

“…They are the best-studied of the non-pathogenic protists and have an established place in the laboratory where they are fairly easily manipulated [13]. They are large enough to image easily with light microscopy and are amenable to routine cytological staining [13,18,19,20]. Staining of surface features for confocal microscopy is a known art, and a functional method was published by Ann Fleury [14,15], Fleury and Laurent [6] and Fleury et al [17].…”

Cytological staining of protozoa: a case study on the impregnation of hypotrichs (Ciliophora: spirotrichea) using laboratory-synthesized protargol

“…Taxonomical and ecological studies on ciliated protozoa have been performed using a number of methods, including live observation, silver carbonate impregnation, scanning electron microscopy, and supravital staining, protargol, and dry and wet silver nitrate impregnation. These techniques have been modified and improved in several studies Lwoff 1930, 1936;Tuffrau 1964Tuffrau , 1967Wilbert 1975Wilbert , 1976Fernandez-Galiano 1976;Foissner 1976Foissner , 1991Foissner , 2014Dieckmann 1995;Silva-Neto 2000;Ma et al 2003;Vdacny and Foissner 2012). Among these methods, protargol impregnation is a key approach used to identify and describe ciliate species (Corliss 1979;Lynn 2008;Foissner 2014).…”