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

Protargol (silver proteinate) impregnation is a common method used to identify and characterize ciliated protozoa. Unfortunately, chemical companies have stopped producing the ‘strong’ protargol powder used in this method. Based on an in-house protocol for its synthesis published in 2013, more than 10 batches of protargol powder were produced and subsequently applied in taxonomic studies. During these studies, the protocol for protargol powder synthesis was slightly modified and employed a peptone not originally listed in the 2013 protocol. This modification improved the results of the impregnation protocol. Protargol preparations of hypotrichs were optimized by adjusting the pH during staining rather than during the synthesis. The pH was adjusted to 7.5–7.6, and an acetone developer was used. While the conditions used in this study are not completely comparable to those using the commercially produced protargol, access to this information could help researchers investigate the diversity of ciliates, particularly hypotrichs.     

四种淡水缘毛类纤毛虫的形态学研究

通过活体显微观察及银染法对采自武汉东湖的4种固着缘毛类纤毛虫: 粗茎睫纤虫Ophrydium crassicaule Penard, 1922, 念珠伪钟虫Pseudovorticella monilata (Tatem, 1870) Foissner & Schiffmann, 1974, 垂盖虫Opercularia nutans (Ehrenberg, 1831) Stein, 1854和一个鞘居虫未定种Vaginicola sp.的活体、纤毛图式、银线系以及细胞核的形态学特征进行详细的描述, 补充和完善了缘毛类纤毛虫的形态学资料, 为纤毛虫分类和系统发育学的研究提供重要信息。此外, 首次在伪钟虫中观察到有性生殖现象, 为纤毛虫生殖进化的研究提供了基础资料。     

Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.     

A practical method of Ludox density gradient centrifugation combined with protargol staining for extracting and estimating ciliates in marine sediments

Methodological impediments have long been the main problem in estimating the ecological role of marine benthic ciliates. Percoll density centrifugation is currently the most efficient technique for extracting ciliates from fine-grained sediments, while the high cost and low density of Percoll limit its wide application. We developed a protocol of density gradient centrifugation using the cheap sol Ludox HS 40 in combination with the quantitative protargol stain (QPS) to enumerate and identify marine benthic ciliates. The combined Ludox-QPS method involves sample collection and salt reduction, extraction with Ludox centrifugation, and preparation with the QPS technique. The recovery efficiency of Ludox was first tested with azoic sandy and muddy sediments. A 94–100% recovery rate of ciliates was reached. The method was further tested with natural sandy, muddy-sand and muddy sediments. Excellent extraction efficiencies were consistently obtained: an average of 97.6% for ciliates in sand, and 96.9–97.8% for nematodes in the three types of sediments. The high efficiencies indicate that the method allows for simultaneous enumeration of micro- and meiobenthos. Advantages of the new method include: (i) reliable and cost-efficient operation; (ii) appropriate centrifugation for both micro- and meiobenthos; and (iii) applicability to large samples and routine ecological surveys.     

西藏三种苔藓栖生纤毛虫的形态和系统发育分析

本研究利用活体观察和蛋白银染色方法对采自西藏日喀则和那曲的3种苔藓栖生纤毛虫,鬃异源棘尾虫(Tetmemena pustulata)、棘毛虫未定种(Sterkiella sp.)和殖口虫未定种(Gonostomum sp.)进行了形态学研究,描述了活体、核器以及纤毛图式等形态学特征.同时,测定了这3种纤毛虫的SSU r...     

西藏温泉两种中国新记录纤毛虫第一双小核草履虫和明布雷斯四膜虫的形态学和系统发育学研究

研究利用活体观察、蛋白银和氨银染色技术对采自西藏温泉的寡膜纲咽膜类纤毛虫第一双小核草履虫(Paramecium primaurelia)和膜口类纤毛虫明布雷斯四膜虫(Tetrahymena mimbres)进行了形态学研究, 首次描述了这两种纤毛虫细胞核器的形态和位置、纤毛图式和口膜的排布模式; 并且测定了SSU rDNA和COXⅠ标记基因序列, 基于这两种基因的系统发育树均支持P. primaurelia聚在草履虫P. aurelia复合种中, T. mimbres聚在四膜虫T. borealis类群中。两种纤毛虫均为中国新记录种, 且首次在高原温泉中发现, 不仅为西藏地区温泉原生动物生物资源的发掘提供新的方法和思路, 也为原生动物环境适应性研究提供基础资料。     

The Stainability of Nerve Fibers by Protargol With Various Fixatives And Staining Technics

The influence of the commonly used tissue fixing reagents, individually and in various combinations, on subsequent staining by protargol was studied. The reagents used were formalin, formamide, picric acid, acetic acid, paranitrophenol, pyridine and chloral hydrate. Parraffin sections from intestine and peripheral nerve of cat, dog, monkey and rat were stained with protargol after fixation in various experimental mixtures of the fixing reagents. Satisfactory nerve stains of intestine were not obtained with regularity after any one fixing and staining procedure. (Good fixation and staining appeared to be influenced by properties inherent in the tissue itself and showed marked variations from animal to animal even in the same species.)Stains of nerve fibers in peripheral nerve trunks were much more easily obtained than in the intestine where good stains were sporadic and unpredictable. The use of a mixture of 0.5% protargol and 0.1% fast green FCF, is proposed as a silver-dye staining medium.     

Identification of the pathogenic ciliate Pseudocohnilembus persalinus (Oligohymenophorea: Scuticociliatia) by fluorescence in situ hybridization

Many scuticociliatid ciliates are regarded as devastating pathogens in aquaculture. Among these, Pseudocohnilembus persalinus is a facultative pathogen that often results in refractory diseases of mariculture fish. Although traditional silver staining methods have been successfully used to identify these ciliates, their identification is hampered by their small size and their morphological similarity to closely related species. We designed an alternative method of identification of P. persalinus using an SSU-rDNA targeted oligonucleotide probe labeled with a fluorochrome, and optimized in a fluorescence in situ hybridization (FISH) protocol. The assay results in a clear identification by strong fluorescence signals from the oligonucleotide probe. The method can be used for quick and early detection of P. persalinus infections on host fish, as well as other susceptible organisms in aquiculture water. It may also be used in studies of the geographical distribution of this scuticociliate.     

Two Anaerobic Ciliates (Ciliophora,Armophorea) from China: Morphology and SSU rDNA Sequence,with Report of a New Species,Metopus paravestitus nov. spec

The morphology and phylogeny of two metopid ciliates, collected from anaerobic habitats in China, were investigated using live observation, protargol staining method, and SSU rDNA sequencing. The new species Metopus paravestitus nov. spec. can be distinguished by a combination of the following features: oblong cell with densely arranged ectobiotic prokaryotes perpendicular to cell surface, filiform intracytoplasmic structures packed in the anterior portion of the cell. Our work also demonstrates the wide geographical distribution of Metopus es (Müller, 1776) Lauterborn, 1916. The order Metopida is consistently depicted as a paraphylum in SSU rDNA phylogeny. Metopus paravestitus nov. spec. is closely related to its marine congeners than to freshwater forms. The present study confirms once again the non-monophyly of the genus Metopus and genus Metopidae.     

土壤腹毛类纤毛虫四新纪录种

利用非淹没培养皿法、活体观察和蛋白银染色法,研究了青岛的土壤纤毛虫,发现2属4种我国新纪录种:澳洲管膜虫(Cyrtohymena australis Foissner,1995)、四核管膜虫[C.quadrinucleata(DragescoNjine,1971)]、犬牙管膜虫[C.candens(Kahl,1932)]及迟钝澳纤虫(Australocirrus oscitans BlattererFoissner,1988)。文中对该4种纤毛虫的活体形态和纤毛图式进行了观察记述,补充了前人描述的不足,并依据统计学特征与相近种群进行了比较。     

松花江四种纤毛虫的形态学研究

利用活体观察, 氨银染色和蛋白银染色的方法, 对采自松花江的4种广布种纤毛虫: 贻贝棘尾虫Stylonychia mytilus (Müller, 1773) Ehrenberg, 1830、鬃异源棘尾虫Tetmemena pustulata (Müller, 1786) Eigner, 1999、尾草履虫Paramecium caudatum (Ehret, 1963)和刚毛榴弹虫Coleps hirtus的形态学进行研究。依据新采集种群, 对4个种活体形态和纤毛图式特征, 以及鬃异源棘尾虫的发生学特征进行补足性描述。研究对贻贝棘尾虫做了重要重描述, 可清晰观察到该种蛋白银染后6列背触毛的排列特征、左右缘棘毛数的具体统计, 结果发现, 贻贝棘尾虫哈尔滨种群与前人报道的种群相比, 形态特征基本吻合, 区别在于前者小膜数目多于后者。鬃异源棘尾虫哈尔滨种群与漠河种群相比, 形态和纤毛图式方面十分相似, 发生模式基本一致, 细微区别在于前者左右缘棘毛数目普遍多于后者, 前者虫体大小波动范围较小, 小核数目多于后者。尾草履虫哈尔滨种群与其青岛种群相比, 二者显著差异在于收集管数目前者比后者少。本文榴弹虫与前人报道的刚毛榴弹虫种群相比, 盔板带的数目、排列方式、窗口类型等形态学基本一致, 细微区别在于本文报道种群体型普遍较前人报道种群略大。     

A Somatic Kinetid Study of the Pycnotrichid Ciliate Vestibulongum corlissi N. G., N. Sp. (Class: Litostomatea), Symbiont in the Intestines of the Surgeonfish,Acanthurus xanthopterus1

ABSTRACT The ciliate Vestibulongum corlissi n. g., n. sp. was collected from the intestines of surgeonfish, Acanthurus xanthopterus, in the summer of 1986. In has been examined in the light microscope after protargol staining and by scanning and transmission electron microscopy. Its form is distinct from that of other pycnotrichid ciliates at the generic level. Somatic kinetids were examined; these demonstrate that its cytostome is posterior and that the kinetid structures and the presence of a second transverse microtubular ribbon confirm its placement in the class Litostomatea.     

Enumeration of small ciliates in culture by flow cytometry and nucleic acid staining

We developed a fast and simple protocol for accurate quantification of small freshwater ciliates by flow cytometry (FCM). The ciliates were stained with several nucleic acid stains such as TO-PRO-1, YO-YO-1 and PicoGreen, and analysed by a commercially available flow cytometer. The method was tested with cultures of the prostomatid species Urotricha farcta and Balanion planctonicum, including the small cryptophyte Cryptomonas sp. as food. Of the dyes tested, TO-PRO-1 gave the best results. Flow cytometric results agreed well with microscopic counts. Due to its greater speed and accuracy, FCM was superior to light microscopy. FCM was also superior to electronical particle counting and sizing (EPCS). Of particular importance, FCM in combination with TO-PRO-1 staining allowed unequivocal discrimination in cases of overlapping size distributions between the target population (i.e., the ciliate predators) and other particles (the cryptophyte prey, detritus).     

广东大亚湾六种海洋刺钩类纤毛虫的形态学研究

利用活体观察及银染法首次对采自广东大亚湾沿岸的盐菲阿虫Phialina salinarum Kahl,1928,海洋长吻虫Lacrymaria marina Kahl,1933,惊扰伪颈毛虫Pseudotrachelocerca trepida(Kahl,1928)Song,1990,贪食纤口虫Chaenea vorax Quennerstedt,1867,柱核柱毛虫Cyclotrichium cyclokaryon Meunier,1907和加冈栉毛虫Didinium gargantua Meunier,1910六种刺钩类纤毛虫进行了形态学研究,补充了活体形态学以及纤毛图式等分类学新信息。首次观察到了海洋长吻虫体纤毛基部的皮层颗粒和柱核柱毛虫大亚湾种群较青岛种群具有更多的体动基列等若干新信息。研究表明,刺钩类纤毛虫在大亚湾近岸水域具有较高的物种多样性。       

一种游仆虫无性分裂生殖的研究Ⅰ.形态学和核器的演化

应用光学显微镜和扫描电子显微镜观察了采自上海的游仆虫的形态,描述了它的表面结构以及纤毛器、棘毛下纤维的结构和核器的演化过程。     

Amphileptus aeschtae nov. spec. and Amphileptus eigneri nov. spec. (Ciliophora, Pleurostomatida), two new marine pleurostomatid ciliates from China

The morphology and infraciliature of two pleurostomatid ciliates, Amphileptus aeschtae nov. spec. and Amphileptus eigneri nov. spec., collected from shrimp-culturing ponds of the Bohai Sea, China, were investigated using live observations and the protargol impregnation method. The new species A. aeschtae is identified by the presence of 200-300 macronuclear nodules and several contractile vacuoles along the ventral side of the cell. A. eigneri differs from its congeners, inter alia, by the marine habitat, the presence of many contractile vacuoles along the ventral margin and the number of somatic kineties on both sides. Loxophyllum ozakii Shigematsu, 1953 is transferred to Kentrophyllum.     

Ciliated protozoa of two antarctic lakes: analysis by quantitative protargol staining and examination of artificial substrates

Planktonic and artificial substrate-associated ciliates have been identified in two perennially ice-covered antarctic lakes of the McMurdo Dry Valleys. Abundances estimated by quantitative protargol staining ranged from <5 to 31690 cells · l⁻¹, levels that are comparable to those previously obtained using other methods. Nineteen ciliate taxa were identified from these lakes, with the most frequently encountered genera being Plagiocampa, Askenasia, Monodinium, Sphaerophrya and Vorticella. The taxonomic findings compare favorably with those of previous investigators; however four previously unreported genera were observed in both Lakes Fryxell and Hoare. The variability in the depth distributions of ciliates in Lake Fryxell is explained in terms of lake physicochemical properties and ciliate prey distributions, while factors related to temporal succession in the Lake Hoare assemblage remain unexplained. Local marine or temperate zone freshwater habitats are a more likely source than the surrounding dry valleys soils for present ciliate colonists in these lakes. Although the taxonomic uncertainties require further examination, our results suggest that ciliate populations in these antarctic lakes undergo significant fluctuations and are more diverse than was previously recognized. Accepted: 27 July 1998     

Description of Deviata rositae n. sp., a new ciliate species (Ciliophora, Stichotrichia) from Argentina

Soil samples were taken from a temporary pond located in Buenos Aires province, Argentina, during the dry phase in the summer of 2005. The ciliates were studied alive and after staining with protargol. Deviata rositae n. sp. measures 112-154 microm in length and 21-28 microm in width in vivo and has a vermiform body. The contractile vacuole is located in the mid-body on the left. The macronucleus is moniliform and there are 1-3 micronuclei. The oral apparatus is composed of 14-18 adoral membranelles and straight paroral and endoral membranes that never intersect each other. The somatic ciliature is arranged in four frontal cirri, one buccal cirrus, six long and slightly spiraled rows of cirri with the first right row extending up to the equatorial or sub-equatorial region, and two dorsal rows of dikinetids. This new species of Deviata primarily differs from its congeners by the number of macronuclear nodules and the number and disposition of the dorsal rows of dikinetids.     

长江口地区五种腹毛类纤毛虫的形态学研究

利用活体观察和蛋白银染色技术,对采自长江口泥滩潮间带及河道的五种腹毛类纤毛虫进行了形态学研究。包括对两个国内新记录种仁川巴库虫Bakuella (Bakuella) incheonensis和希斯多利织毛虫Histriculus histrio提供了详细的活体特征和纤毛图式信息,对红色伪角毛虫Pseudokeronopsis rubra、黄色伪角毛虫Pseudokeronopsis flava和冠突伪尾柱虫Pseudourostyla cristata进行了形态学重描述。五种腹毛类纤毛虫的长江口种群与国内外种群均存在不同程度的形态学差异:仁川巴库虫较韩国原始种群体型大,口围带小膜和额前棘毛数较多;希斯多利织毛虫与部分国外种群存在体长差异;红色伪角毛虫与青岛种群相比形态特征基本吻合但个体大小的波动范围较大;黄色伪角毛虫与湛江种群相比额棘毛数目较多;冠突伪尾柱虫较奥地利种群体型较小,与日本种群相比大核较多。该工作丰富了中国腹毛类纤毛虫多样性的认识。