Protargol Staining Research Articles

Morphogenesis and molecular phylogeny of the soil ciliate Rigidohymena quadrinucleata (Dragesco and Njine, 1971) Berger, 2011 (Ciliophora, Hypotricha, Oxytrichidae)

Morphogenesis in the soil hypotrich ciliate, Rigidohymena quadrinucleata (Dragesco and Njine, 1971) Berger, 2011; was investigated using protargol staining. The main events during binary fission are as follows: (1) the proter retains the parental adoral zone of membranelles entirely, whereas the old undulating membranes dedifferentiate into an anlage that gives rise to the leftmost frontal cirrus and the new undulating membranes of the proter; (2) six streaks of frontoventral-transverse cirral anlagen are segmented in the pattern 1:3:3:3:4:4 from left to right, which form three frontal, four frontoventral, one buccal, five ventral and five transverse cirri, respectively; (3) the postoral ventral cirrus V/3 is not involved in primordia formation demonstrating that this species belongs to the stylonychines; (4) marginal anlagen are formed intrakinetally and the right marginal anlagen develop much earlier than the left ones; (5) the generation of the dorsal kineties is of typical Oxytricha-pattern and three caudal cirri are formed, one at the posterior end of each of dorsal kineties 1, 2 and 4. Phylogenetic analyses based on SSU rDNA sequence data indicate that Rigidohymena is polyphyletic and that R. quadrinucleata clusters with R. candens with full support.

Description and Molecular Phylogeny of a Novel Hypotrich Ciliate from the Soil of Marche Region, Italy; Including Notes on the MOSYSS Project.

The morphology and morphogenesis during cell division of a new stylonychine hypotrich, Rigidocortex quadrinucleatus n. sp., were investigated using live observation and protargol staining. The new species was isolated from soil samples collected from an organic farm in the Marche Region, Italy, in framework of the MOSYSS project. Rigidocortex quadrinucleatus is characterized as follows: cell size about 180×80μm invivo; four ellipsoidal macronuclear nodules; 44 adoral membranelles: 18 fronto-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse, and five transverse cirri; dorsal kinety 3 with multiple fragmentation; resting cyst with hyaline ridges. Rigidocortex quadrinucleatus mainly differs from the type species R.octonucleatus in having four (vs. eight) macronuclear nodules. Rigidocortex quadrinucleatus can be easily confused with Sterkiella cavicola since both have a rather similar ventral ciliature; however, they can be separated by the slightly higher number of cirri in the left marginal row that runs along the posterior cell's margin in R.quadrinucleatus. Morphogenesis on the ventral surface is highly similar to that of Sterkiella species, but differs significantly on the dorsal surface (multiple vs. simple fragmentation of dorsal kinety 3). Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to S.cavicola.

Aviisotricha hoazini n. gen., n. sp., the Morphology and Molecular Phylogeny of an Anaerobic Ciliate from the Crop of the Hoatzin (Opisthocomus hoazin), the Cow Among the Birds

The hoatzin is the only known avian species that has evolved a foregut fermentation system similar to that of ruminant animals. Due to the closeness of the bird's fermentation chamber, the crop, to the bird's beak it exudes an unpleasant odour; therefore, the hoatzin is called the "cow among the birds". In addition to Eubacteria and Archaea, responsible for digestion of the vegetation they consume, the bird's crop contains a holotrich ciliate, described here for the first time in detail. Cytological staining of this isotrichid-like ciliate with the Chatton-Lwoff and Protargol staining procedures, as well as SEM and TEM, justified the establishment of the new genus Aviisotricha n. gen. with its new type species Aviisotricha hoazini n. gen., n. sp.. Phylogenetic analyses of a portion of the small subunit rRNA gene supported the taxonomic placement of this new genus and species in the family Isotrichidae. Aviisotricha is compared with Balantidium, Dasytricha and Isotricha with special reference to their dorsal brushes, which show similarity to the paralabial organelle of the Entodiniomorphida. The possible phylogenetic origin of Aviisotricha is discussed and a taxonomic revision of the family Isotrichidae is given.

Description of a New Brackish Water Ciliate, Uronychia xinjiangensis n. sp. (Ciliophora, Euplotida) Based on Morphology, Morphogenesis and Molecular Phylogeny

A brackish water euplotid ciliate, Uronychia xinjiangensis n. sp., was discovered in a ditch in Yuli County, Xinjiang, China. Its morphology, ciliature and morphogenesis were investigated based on specimens examined in vivo and following protargol staining. The new species is characterized by the posterior part of the adoral zone composed of three membranelles, which has never been seen in all other known congeners. Other morphologic features include: (i) body oval-shaped, with conspicuous right anterior spur-like protrusion; (ii)size in vivo 60–90 × 40–68 μm; (iii) two macronuclear nodules; (iv) four frontal, two ventral, five transverse, three left marginal and three caudal cirri. Its morphogenesis proceeds in a usual way, except that the oral primordium forms only three proximal membranelles rather than four proximal membranelles within congeners. The small subunit rRNA gene of the new species (GenBank accession number: KX147287) comprises 1723 bp and 44.63% GC content, and differs by 0.12–1.81% from those of congeners. Phylogenetic analyses based on SSU rRNA gene sequence data reveal that Uronychia xinjiangensis n. sp. clusters with other Uronychia species with full support, which supports the monophyly of the genus Uronychia Stein, 1859.

Rigidosticha italiensis n. gen., n. sp. (Ciliophora, Spirotricha), a novel large hypotrich ciliate from the soil of Lombardia, Italy

The morphology of Rigidosticha italiensis n. gen., n. sp., which was found in a soil sample collected from an uncultivated field in Lombardia, Italy, was investigated using live observation and protargol staining. Rigidosticha n. gen. is characterised by a rigid body, undulating membranes resembling a Steinia pattern, oxytrichid frontal ciliature, distinct mid-ventral cirral pairs, transverse cirri, one right and one left row of marginal cirri, absence of dorsal kinety 3 fragmentation, more than two dorsomarginal rows, and caudal cirri. The new species shows the following features: size in vivo 230–330×100–170μm, on average 230×115μm in protargol preparations; two ellipsoidal macronuclear nodules; 51 adoral membranelles; one buccal cirrus; one parabuccal cirrus; two frontoterminal cirri; 16 mid-ventral cirral pairs; and three transverse cirri. Rigidosticha mainly differs from Rigidothrix, Afrophrya, Uroleptus, and Territricha, in having the undulating membranes in Rigidosticha (vs. oxytrichid and cyrtohymenid) pattern. The oxytrichid frontal ciliature and midventral pattern in the present species further support the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis.

Morphology, Ontogeny, and Molecular Phylogeny of Two Freshwater Species of Deviata (Ciliophora, Hypotrichia) from Southern China.

The morphology and partial morphogenesis of two freshwater hypotrichous ciliates, Deviata brasiliensis Siqueira-Castro etal., 2009 and Deviata rositae Küppers etal., 2007, isolated from southern China, were investigated using live observation and protargol staining. Our populations resemble the original ones in terms of their live characters and ciliary patterns. The main determinable morphogenetic features of D.brasiliensis basically correspond with those of the type population. However, the origin of anlage V for either proter or opisthe is ambiguous: whether anlage V for the proter originates from parental frontoventral row 2 (the same as in the original population) or parental frontoventral row 3 (the same as in Deviata abbrevescens) or even de novo is not clear; the anlage V for the opisthe is possibly derived from frontoventral row 3 and further migrates to frontoventral row 2, like that in D.abbrevescens. In addition, the SSU rRNA gene was first sequenced for both species. Molecular phylogenetic analyses suggest that the genus Deviata is non-monophyletic and has a close relationship with Perisincirra paucicirrata.

Redescription of two little-known urostyloid ciliates, Anteholosticha randani ( ) and A. antecirrata (Ciliophora, Urostylida)

The morphology of two little-known urostyloid ciliates, Anteholosticha randani (Grolière, 1975) Berger, 2003 and A. antecirrataBerger, 2006, collected from freshwater biotopes in southern China, was studied based on live observations and protargol staining. Anteholosticha randani is characterized by its bipartite adoral zone and short, longitudinally aligned undulating membranes. One early stage of reorganization/morphogenesis, one early-middle stage of reorganization and one middle stage of morphogenesis are also reported. Anteholosticha antecirrata is characterized by its large body size in vivo (200–400×40–80μm), a row of buccal cirri and conspicuous, yellow-green cortical granules. Phylogenetic analyses based on SSU rDNA sequence data reveal that A. antecirrata may share a most recent common ancestor with Urostyla grandis and Bakuella granulifera, whereas A. randani branches independently and is sister to a large clade that includes Pseudourostyla, Pseudokeronopsis, Caudiholosticha and several species of Anteholosticha.

Morphology and systematics of two freshwater urostylid ciliates, with description of a new species (Protista, Ciliophora, Hypotrichia)

The morphology of two freshwater urostylid species, Neourostylopsis flava spec. nov. and Pseudourostyla subtropicaChen et al., 2014, isolated from freshwater ponds in northern and southern China, respectively, was investigated following examination of specimens in vivo and following protargol staining. Neourostylopsis flava spec. nov. is distinguished from its congeners by the following characteristics: body size 150–220×50–75μm in vivo; yellow in colour; bright yellow to yellow-brownish spherical cortical granules densely arranged along marginal cirral rows and in irregular short rows on dorsal side; adoral zone with 40–55 membranelles; six to eight frontal, three or four buccal, two pretransverse ventral and seven to nine transverse cirri; 27–40 midventral pairs extending to about anterior 55% of cell; four or five left and four right marginal rows; freshwater habitat. A redescription of a freshwater population of P. subtropica is also provided. Phylogenetic analyses based on small subunit ribosomal DNA sequences shows that P. subtropica and N. flava spec. nov. group with their congeners and both Neourostylopsis and Pseudourostyla are monophyletic.

Taxonomic Redescription of Loxophyllum perihoplophorum and L. rostratum (Ciliophora: Pleurostomatida) from Korea

Two pleurostomatid ciliates, Loxophyllum perihoplophorum Buddenbrock, 1920 and L. rostratum Cohn, 1866, were collected from the coastal waters of the East Sea, Korea. Their morphologies are described based on live observation and protargol staining, and morphometrics are provided. Loxophyllum perihoplophorum is characterized by the following features: 200-650 μm long in vivo; body slender leaf-shaped, flexible and contractile, with thin and wide extrusome-belted zone; 2 macronuclear nodules (Ma) and 1 micronucleus (Mi); 7-9 contractile vacuoles (CV) positioned along dorsal margin; extrusomes (Ex) evenly distributed along edge of entire body, with about 10 dorsal warts (Wa); 9-11 left (LSK) and 19-22 right somatic kineties (RSK), 4-5 furrows (Fu) on left side. Loxophyllum rostratum is about 100-130 μm long in vivo; body oblate leaf-shaped, contractile, convex ventral side and S-shaped dorsal side, beaklike anterior end; 2 Ma and 1 Mi; 1 CV terminally located; Ex distributed along edge of entire body, with about 9-10 dorsal Wa; 7-8 LSK and 15-19 RSK, ca. 5 Fu on left body side. In addition, sequences of small subunit ribosomal DNA were determined from these two Loxophyllum species and compared with the known Loxophyllum sequences.

Description and phylogenetic position of Corlissina maricaensis gen. nov., sp. nov. (Karyorelictea, Geleiidae), a novel interstitial ciliate from Brazil, with redefinition of the family Geleiidae

Corlissina maricaensis gen. nov., sp. nov. was obtained from samples of sediment collected in a brackish lagoon of Maricá city, Rio de Janeiro state, Brazil. The morphological description was based on live observations, after protargol staining, and scanning electron microscopy. The novel species has a cylindrical body shape that is slightly contractile, 230-550 × 35-65 μm, a cytoplasm with many globular inclusions, one row of irregular cortical granules between each somatic kinety, approximately 40-62 somatic kineties, two globular macronuclei measuring 9-24 μm and one micronucleus of approximately 4-9 μm. A subapical oral cavity was approximately 20-80 × 9-25 μm, with an adoral zone on the left side of the buccal field, which was composed of 32-60 polykineties and a paroral at the right side that was composed of 40-57 short polykineties. The new genus is distinguished from other geleiids by a loop-shaped posterior end of the paroral ciliature, made up of two rows of short polykineties, and the oralization of the central superior kinety (K0i), forming a row of dikinetids that borders the adoral zone internally, followed by several rows of monokinetids. In the phylogenetic analyses, the novel species was recovered as the sister group of Parduczia orbis with full support values based on 18S rRNA gene sequences. This work also indicates some problems in the definitions of the Geleiidae and proposes a new diagnosis for this karyorelictid family.

Novel Discovery of Two Heterotrichid Ciliates, Climacostomum virens and Fabrea salina (Ciliophora: Heterotrichea: Heterotrichida) in Korea

Two heterotrichid ciliates, Climacostomum virens (Ehrenberg, 1838) Stein, 1859 from brackish water and freshwater, and Fabrea salina Henneguy, 1890 from a solar saltern, were collected in Korea. They are novelly investigated in Korea by means of live observation, protargol staining and nuclear small subunit (SSU) rRNA gene sequencing. Climacostomum virens is characterized by pouch-like body shape, body length of <TEX>$200-370{\mu}m$</TEX> in vivo, conspicuous cytopharyngeal tube, macronuclei ribbon-like shape, and one to four in number, with or without symbiont algae in cytoplasm, 34-66 somatic kineties, 67-113 adoral zone of membranelles, 8-42 peristomial kineties, 24-37 apical membranelles. SSU rDNA sequence size is 1,591 bp and GC contents 48.52%. Fabrea salina is also characterized by scoop-like body shape with proboscis, body length of <TEX>$190-240{\mu}m$</TEX> in vivo, one to two rod-shaped macronuclei, oval micronuclei, grayish green cortical granules, 104-186 somatic kineties, 4-8 preoral kineties, 7-19 peristomial kineties and fragmented paroral membrane. SSU rDNA sequence size is 1,598 bp and GC contents 47.50%.

Taxonomy and molecular phylogeny of two novel ciliates, with establishment of a new genus, Pseudogastrostyla n. g. (Ciliophora, Hypotrichia, Oxytrichidae)

Two novel hypotrichous ciliates, Pseudogastrostyla flava n. g., n. sp. and Urosomoida subtropica n. sp., isolated from Daya Bay Mangrove Park, Huizhou, southern China, were observed in vivo and after protargol staining. Pseudogastrostyla n. g. is diagnosed by having more than 18 frontal-ventral-transverse cirri, an indistinct frontoventral row, undulating membranes in Oxytricha-pattern and dorsomarginal kinety, but lacking dorsal kinety fragmentation during morphogenesis. The monotypic species, P. flava n. sp., is characterized by its brownish body colour, brown-yellowish cortical granules mostly arranged in groups, frontoventral row terminating near mid-body, and one caudal cirrus. Urosomoida subtropica n. sp. can be recognized by having one micronucleus located between two macronuclear nodules, four transverse cirri, pretransverse ventral cirrus (V/2) anteriorly located, four dorsal kineties and three caudal cirri. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that Pseudogastrostyla n. g. is nested within the oxytrichids and is most closely related to Rubrioxytricha ferruginea, and that Urosomoida subtropica n. sp. clusters with Oxytricha elegans rather than the type species, U. agilis. Nodal support values are, however, too low to draw any conclusions about the molecular systematics of the genus Urosomoida.

Methods for taxonomic studies of rumen ciliates (alveolata: ciliophora): a brief review.

This review presents the principal methods used in taxonomic studies of rumen ciliates: live observation, Lugol staining, fixation and staining with methyl-green formalin saline (MFS) solution, protargol staining, silver carbonate impregnation, scanning electron microscopy and molecular techniques. Mastering these techniques is essential for successful research on the taxonomy of rumen ciliates. No single technique reveals all of the characteristics required for a complete description of a rumen ciliate; therefore, it is necessary to combine the use of these techniques as appropriate to the rumen ciliate group under study. Tables are provided to summarize: 1) morphological methods more appropriate for revealing morphological structures of interest, 2) morphological methods indicated for each group of rumen ciliates, and 3) main primers used for PCR amplification of the 18S rDNA of rumen ciliates.

Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India

The morphology and morphogenesis during cell division of Sterkiella tetracirrata n. sp., isolated from a soil sample collected from the Silent Valley National Park, Kerala, India, were investigated using live observation, protargol staining and scanning electron microscopy. The new species differs from its congeners by the following combination of features: cell size in vivo 85–110×35–50μm, on average 84×37μm in protargol preparations; four ellipsoidal macronuclear nodules; 31 adoral membranelles; 17 frontal-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse and invariably four transverse cirri; resting cyst with separate macronuclear nodules. Sterkiella tetracirrata differs from the similar species S. terricola in the number of transverse cirri (invariably 4 vs. 3) and in the number of adoral membranelles (24–35 vs. 22 or 23). Morphogenesis resembles that of its congeners S. nova and S. histriomuscorum. Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to either S. cavicola or S. histriomuscorum. The analyses support the morphological evidence (e.g., similarity in the oral apparatus and the dorsal kinety pattern) that Gastrostyla and Pattersoniella evolved from a Sterkiella-like ancestor.

Phylogenetic position of the genus Cyrtostrombidium, with a description of Cyrtostrombidium paralongisomum nov. spec. and a redescription of Cyrtostrombidium longisomum Lynn & Gilron, 1993 (Protozoa, Ciliophora) based on live observation, protargol impregnation, and 18S rDNA sequences.

We redescribe Cyrtostrombidium longisomum Lynn & Gilron, 1993, the type species of the genus Cyrtostrombidium, and describe the new species Cyrtostrombidium paralongisomum n. sp. using live observation, protargol staining and molecular data. The morphological characters of these two species are clearly distinct, i.e., dikinetid numbers in the girdle and ventral kineties; however, it is difficult to separate them by 18S rDNA sequences because they differ by only 8 bp, indicating that 18S rDNA sequences are insufficient for separating different species in the genus Cyrtostrombidium. We not only observed the position of the oral primordium in the genus Cyrtostrombidium but also observed a possibly homoplasious trait, a dorsal split in the girdle kinety, in (1) Apostrombidium, (2) Varistrombidium, and (3) Cyrtostrombidium/Williophrya. This partially supports the hypothesis of somatic ciliary pattern evolution recently put forth by Agatha and Strüder-Kypke.

Morphology, morphogenesis and molecular phylogeny of an oxytrichid ciliate, Rubrioxytricha haematoplasma (Blatterer & Foissner, 1990) Berger, 1999 (Ciliophora, Hypotricha).

The morphology and morphogenesis of an oxytrichid ciliate, Rubrioxytricha haematoplasma (Blatterer & Foissner, 1990) Berger, 1999, collected from brackish and marine waters in China, were investigated using live observation and the protargol staining method. The main features of the morphogenetic process are: (i) the parental adoral zone of membranelles is retained completely in the proter and the anlage of undulating membranes originates from dedifferentiation of the old structures; (ii) three frontal, four frontoventral, one buccal, five ventral and five transverse cirri are derived from the anlagen of the undulating membranes and the five streaks of frontal-ventral-transverse anlagen in the pattern of 1:3:3:3:4:4 from left to right; (iii) the morphogenesis of the dorsal kineties is simpler than the Oxytricha pattern, i.e. without fragmentation of the dorsal kinety 3 anlagen; (iv) the single caudal cirrus originates from the dorsal kinety 3 anlage on the right side; (v) the two macronuclear nodules fuse into a single mass during the mid-stage of morphogenesis. These features correspond well with Rubrioxytricha indica, indicating that the morphogenetic pattern of Rubrioxytricha is stable. Phylogenetic analysis based on small-subunit rRNA gene sequence data supports the monophyly of the genus Rubrioxytricha, which is nested within the non-Stylonychinae clade.

Phylogeny of the Poorly Known Ciliates, Microthoracida, a Systematically Confused Taxon (Ciliophora), with Morphological Reports of Three Species

Three species of Microthoracids, Lopezoterenia paratorpens n. sp., Trochiliopsis australis Foissner et al., 1988 and Discotricha papillifera Tuffrau, 1954, collected from Chinese coastal waters, were investigated using live observation and protargol staining methods. Lopezoterenia paratorpens n. sp. was characterized by its squarely shaped cortical papillae and by dorsal kineties which contained loosely distributed basal bodies. Trochiliopsis australis was revealed to have two oral membranelles, which was not recorded in the original report. Phylogenetic analyses were carried out based on SSU rRNA gene sequence data from each of the three species, and on other available data for microthoracids. The results showed that the order Microthoracida is not monophyletic because the family Discotrichidae, which contains L. paratorpens and D. papillifera, forms a clade separated from the "core" Microthoracids clade. The topologies of the maximum likelihood and Bayesian inference trees, along with the distinct morphological characteristics found previously, suggest that the family Discotrichidae should not be assigned to the order Microthoracida. We propose to designate a new order, Discotrichida n. ord. which diagnosed as: flattened ciliates with conspicuous cortical papillae on both dorsal and ventral faces, rod-shaped mucocysts, and an asymmetric cytopharyngeal basket. Also, the fact that Leptopharynx clusters with the assemblage including T. australis, and Pseudomicrothorax is located distantly from Leptopharynx indicates that the classification of Pseudomicrothoracidae and Microthoracidae by Foissner (1985) is justified.

Phylogeny of the Poorly Known Ciliates, Microthoracida, a Systematically Confused Taxon (Ciliophora),with Morphological Reports of Three Species.

Three species of Microthoracids, Lopezoterenia paratorpens n. sp., Trochiliopsis australis Foissner etal., 1988 and Discotricha papillifera Tuffrau, 1954, collected from Chinese coastal waters, were investigated using live observation and protargol staining methods. Lopezoterenia paratorpens n. sp. was characterized by its squarely shaped cortical papillae and by dorsal kineties which contained loosely distributed basal bodies. Trochiliopsis australis was revealed to have two oral membranelles, which was not recorded in the original report. Phylogenetic analyses were carried out based on SSU rRNA gene sequence data from each of the three species, and on other available data for microthoracids. The results showed that the order Microthoracida is not monophyletic because the family Discotrichidae, which contains L. paratorpens and D. papillifera, forms a clade separated from the "core" Microthoracids clade. The topologies of the ML and BI trees, along with the distinct morphological characteristics found previously, suggest that the family Discotrichidae should not be assigned to the order Microthoracida. We propose to designate a new order, Discotrichida n. ord. which diagnosed as: flattened ciliates with conspicuous cortical papillae on both dorsal and ventral faces, rod-shaped mucocysts, and an asymmetric cytopharyngeal basket. Also, the fact that Leptopharynx clusters with the assemblage including T. australis, and Pseudomicrothorax is located distantly from Leptopharynx indicates that the classification of Pseudomicrothoracidae and Microthoracidae by Foissner (1985) is justified. This article is protected by copyright. All rights reserved.

Planktonic ciliate community structure in shallow lakes of lowland Western Europe

Temperate shallow meso- to eutrophic lakes can exist in one of two alternative states with contrasting foodwebs, referred to as the clear-water and the turbid state. We describe the planktonic ciliate communities of such lakes based on a survey of 66 northwestern European lakes. Ciliates were enumerated and identified to species level according to the quantitative protargol staining technique. Ciliate biomass was on average twice as high in the turbid than in the clear-water lakes. The ciliate communities were dominated by oligotrichs and protostomatids, and no differences in functional composition or α-diversity could be detected between turbid and clear-water lakes, although β-diversity tended to be higher in the latter. At the species level, however, community structure strongly differed between turbid and clear-water lakes, and several indicator species could be identified for the different lake categories. Variation partitioning showed that nutrient status did not explain ciliate community structure independent of the alternative states, while lake area was identified as an additional structuring factor for the ciliate communities. These results stress the importance of the ecosystem structure in shaping ciliate communities in temperate shallow lakes and suggest that nutrient status has little direct effect on ciliate community structure in such lakes.

Protargol Synthesis: An In‐house Protocol

The protargol staining method has proved to be indispensable for revealing the cellular structures of a variety of protozoa, especially the flagellates and ciliates. Protargol provides permanent stains of a variety of cellular structures: nuclei, extrusomes, basal bodies, and microfilamentous constituents of cells. Together with the older silver nitrate methods, protargol impregnations have provided the basis for the detailed descriptions of nearly all ciliates to date. The performance of commercially available preparations has varied widely. Recently, suppliers have stopped stocking the effective forms of protargol resulting in a worldwide shortage. Thus, it has become necessary for protistologists to explore on-site synthesis of this critically important agent. An optimum protocol for synthesis of protargol should be rapid, relatively inexpensive, simple enough to be done by non-chemists, and achievable without specialized equipment. In this article, the authors briefly review the interesting history of protargol and describe a protocol, based on the early studies of neuroanatomists, that yields a protargol producing impregnations of ciliates comparable to those obtained with previously available commercial preparations.