An Optimized Protocol of Protargol Staining for Ciliated Protozoa

Protargol staining is a crucial method to reveal the infraciliature of ciliates, which is the most important morphological character for species identification. In the present study, Wilbert's protocol of protargol staining was emended mainly toward the highly happened improper bleaching. Through reciprocal treatments, both insufficient and excessive bleachings were much eliminated from the protargol protocol and the tests performed with four different species of ciliates established that the stainings were considerably improved and more reliable with optimized bleaching. Compared to the original protocol, the optimized method was proved to be more suitable for the groups difficult to stain, and it is also friendlier for the beginners and researchers in related fields.     

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.     

卡龙游仆虫无性生殖期间的形态发生学研究：原生动物,纤毛虫

卡龙游仆虫为海洋中自由生纤毛虫,利用银染法对该种二分裂期间的形态发生学进行了初步的研究,其主要过程为：１．伴随大核改组带的出现和ＤＮＡ复制开始,口原基发生于老口围 方皮膜下一龛腔内,后由前至后组装成围口小膜而演化为后ＡＺＭ。老口围带及口侧膜在原位被被前仔虫继承;２．体棘毛场首先出现两组棘毛原基,其随后各自独立演化成９根前、后仔虫的额－腹－横棘毛;３．缘棘毛原基也为独立发生,初为单一,后断裂为二并分     

Preparation and Test In G Of Silver-Protein Compounds

Silver derivatives of the protargol type were made from 13 different commercial peptones. The peptone samples were purified by precipitating with an ethanol concentration of 75%, reprecipitating once, and discarding any water-insoluble material. A 20-22% solution of the purified material in water, allcalinized with 2 ml. of strong ammonia per 100 ml., was precipitated by adding an equal volume of 25% aqueous AgNO₃, and stood in a refrigerator overnight. Thorough washing of the precipitate with distilled water, draining, and then dissolving it in a purified peptone solution (30-35% aqueous), whose volume was 0.6 that of the solution used for silver precipitation, gave the soluble silver derivative. Ammonia was added to facilitate solution and the final pH adjusted to 8.0-8.4. The concentrated solution was dehydrated either with acetone or in a dessicator under reduced pressure and ground to a powder. Staining tests on neurological tissue showed that the Pharmaceutical peptone (Cudahy Packing Co., Omaha, Nebr.) and the Bacteriologic peptone (Wilson Laboratories, Chicago, 111.) gave the best preparations for staining nerve fibers, and that these were essentially equal to the formerly available German made Protargol.     

The edaphic quantitative protargol stain: a sampling protocol for assessing soil ciliate abundance and diversity

It has been suggested that species loss from microbial groups low in diversity that occupy trophic positions close to the base of the detrital food web could be critical for terrestrial ecosystem functioning. Among the protozoans within the soil microbial loop, ciliates are presumably the least abundant and of low diversity. However, the lack of a standardized method to quantitatively enumerate and identify them has hampered our knowledge about the magnitude of their active and potential diversity, and about the interactions in which they are involved. Thus, the Edaphic Quantitative Protargol Staining (EQPS) method is provided to simultaneously account for ciliate species richness and abundance in a quantitative and qualitative way. This direct method allows this rapid and simultaneous assessment by merging the Non-flooded Petri Dish (NFPD) method [Prog. Protistol. 2 (1987) 69] and the Quantitative Protargol Stain (QPS) method [Montagnes, D.J.S., Lynn, D.H., 1993. A quantitative protargol stain (QPS) for ciliates and other protists. In: Kemp, P.F., Sherr, B.F., Sherr, E.B., Cole, J.J. (Eds.), Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton, FL, pp. 229-240]. The abovementioned protocols were refined by experiments examining the spatial distribution of ciliates under natural field conditions, sampling intensity, the effect of storage, and the use of cytological preparations versus live observations. The EQPS could be useful in ecological studies since it provides both a "snapshot" of the active and effective diversity and a robust estimate of the potential diversity.     

海洋纤毛虫--水滴伪康纤虫的口器发生及形态学重描述

利用蛋白银法对采自山东胶州育虾池的一种海洋盾纤类纤毛虫,水滴伪康纤虫(Pseudocohnilembus persalinus Evans&Thompson,1964)的口器发生过程进行了详细的观察和研究,并对其形态学做了补足性描述。文章通过对该青岛种群发生过程的研究,认为前人所报道的种群(Evans&Thompson,1964;Pomp&Wilbert,1988)缺乏对某个发生关键时期的观察而存在着错误,即:后仔虫的小膜2明确来自老的口侧膜,而不是前人报道的盾片。此外,文章还发现该种的发生与本属另一哈氏伪康纤虫的发生过程几乎完全相同。主要细胞发生过程为:盾片最先增殖,形成初级原基区,然后分裂成前后两部分,前部分最终消失,而后部分最终形成后仔虫的小膜3。继盾片增殖之后口侧膜的锯齿状结构沿细胞纵轴方向分裂成两列,右侧的一列增殖形成次级原基区,之后分裂成前后两部分,前部分迁移形成后仔虫的口侧膜和盾片,后部分形成后仔虫的小膜1和小膜2;老口侧膜的残余部分形成前仔虫的口侧膜及盾片。老的小膜1、小膜2和小膜3则完全为前仔虫所继承。     

Modification of the Silver Proteinate Impregnation Technique for Protozoa and Cultured Nerve Cells

Silver impregnation with silver-protein compounds is widely used for staining tissue sections and cell cultures. Some authors report that the results obtained with these methods have not always been reproducible because the reagent's composition varies according to the manufacturer. To avoid this problem in the method described in this paper, a silver proteinate, produced in our own laboratory is used. Although our method is based on Bodian's, the modifications we have made allows its use for both free-living cells (protozoa) and cells grown in culture (nerve cells). The significant modifications are 1) different fixation, 2) postfixation with Cajal's for-mol-bromide, 3) changes in the duration of the impregnation steps technique and 4) elimination of metallic copper. The method reported here enables us to use silver proteinate whenever we require it and to control the composition of the silver proteinate. This technique can be used for cells cultured in either plastic or glass.     

Staining Sections of Peripheral Nerves for Axis Cylinders and for Myelin Sheaths

Pieces of mammalian nerves 1 to 2 cm. long were placed under moderate tension and fixed 24–48 hours in: picric acid, saturated aqueous, 90 ml.; formalin, 10 ml.; and trichloracetic acid, 25% aqueous, 2 ml. They were washed in water, cut in two and one end stained with 0.04–0.06% osmic acid solution, while the other was dehydrated, embedded in paraffin, and mounted sections from it stained with protargol. The fixing solution used was selected from a number of combinations of acidified picro-formalin as the one most likely to give satisfactory results when followed by both silver and osmic acid. The use of osmic acid solutions of less than 0.1% concentration avoided the overstaining of myelin sheaths seen frequently when stronger solutions were used with material that had been fixed previously. Protargol, 0.5% solution with fast green FCF added to make 0.05% dye in the final concentration, was used to impregnate sections for axis cylinders. Reduction and toning were done as in Bodian's method.     

Staining Sections of Peripheral Nerves for Axis Cylinders and for Myelin Sheaths

Pieces of mammalian nerves 1 to 2 cm. long were placed under moderate tension and fixed 24-48 hours in: picric acid, saturated aqueous, 90 ml.; formalin, 10 ml.; and trichloracetic acid, 25% aqueous, 2 ml. They were washed in water, cut in two and one end stained with 0.04-0.06% osmic acid solution, while the other was dehydrated, embedded in paraffin, and mounted sections from it stained with protargol. The fixing solution used was selected from a number of combinations of acidified picro-formalin as the one most likely to give satisfactory results when followed by both silver and osmic acid. The use of osmic acid solutions of less than 0.1% concentration avoided the overstaining of myelin sheaths seen frequently when stronger solutions were used with material that had been fixed previously. Protargol, 0.5% solution with fast green FCF added to make 0.05% dye in the final concentration, was used to impregnate sections for axis cylinders. Reduction and toning were done as in Bodian's method.     

Morphology and Morphogenesis of Onychodromus quadricornutus n. sp. (Ciliophora,Hypotrichida), an Extraordinarily Large Ciliate with Dorsal Horns1

The morphology and the morphogenesis of the freshwater hypotrich ciliate Onychodromus quadricornutus n. sp. have been investigated using living organisms, protargol impregnation, and scanning electron microscopy. Some preliminary and supplementary results about the morphogenesis of O. grandis and Laurentiella acuminata are included. The new species is unique among all described hypotrichs in having four dorsal horns, whose function is unknown. In addition, O. quadricornutus is probably the most voluminous hypotrich ciliate known (2 times 10^-6-5 times 10⁶μm³). Its morphogenetic pattern resembles the oxytrichids O. grandis and L. acuminata. The strongest apomorphic character, which unites these three species, is probably the multiple fragmentation of the dorsal primordia during morphogenesis. This fragmentation causes the characteristic high number and more or less irregular distribution of the dorsal kineties in the non-dividing individuals.     

Morphology and Morphogenesis of Strobilidium caudatum (Fromentel), Meseres corlissi N. Sp., Halteria grandinella (Müller), and Strombidium rehwaldi N. Sp., and a Proposed Phylogenetic System for Oligotrich Ciliates (Protozoa,Ciliophora)1

ABSTRACT. The morphology and morphogenesis of some oligotrichs were investigated using protargol impregnation, silver carbonate impregnation and scanning electron microscopy. The somatic kineties of Strobilidium caudatum form a spiral at the posterior pole. Strobilidiids without such a spiral are transferred to the genus Rimostrombidium. Fourteen new combinations and a nomen novum, Strobilidium kahli, are necessary, Meseres corlissi n. sp. is characterized by eight somatic kineties composed of long cilia which are not fused to “bristles” as they are in Halteria. Strombidium oblongum shows similar characteristics and is thus combined with Meseres. Strombidium rehwaldi n. sp. has an anterior and an equatorial girdle of extrusomes. The morphogenesis of Meseres and Halteria is very similar, i.e. the entire somatic ciliature and the oral primordium originate apokinetally on the cell surface; the parental somatic ciliature is resorbed. In strobilidiids and tintinnids, the oral anlagen develop in a subsurface pouch and the parental somatic kineties, which are not resorbed, elongate by intrakinetal proliferation of basal bodies. In strombidiids, the oral primordium develops in an intracellular sac or tube. These morphogenetic peculiarities and distinct morphologic characters (e.g. arrangement of adoral membranelles) were applied in constructing a phylogenetic system for oligotrichs using hypotrichs as outgroup. This shows that halteriids are more closely related to hypotrichs than they are to other oligotrichs. The Halteriidae are thus raised to ordinal and subclass ranks, Halteriida n. ord., Halteriia n. subcl.     

海洋尾丝虫无性生殖期间口器发生的再研究

利用蛋白银技术研究了海洋纤毛虫—海洋尾丝虫无性生殖期间的口器发生过程。结果显示其口器发生与已知的同属种类具相似的过程和形式。其口器发生及演化的基本模式可表示如下 :后仔虫的小膜 1、小膜 2来源于老口侧膜后段的增殖 ;后仔虫的口侧膜及盾片来源于老口侧膜前段的增殖 ;后仔虫的小膜 3来自于老口区盾片的增殖 ;前仔虫的口侧膜及盾片来源于老口侧膜 ,三片小膜在口器发生过程中被保留。文中根据现有的形态发生资料对尾丝虫科的系统关系进行了探讨     

A Combination Staining Method for Fibers and Cell Bodies of the Urodele Central Nervous System

A simple method for staining nerve cells and fibers of the salamander central nervous system is described. The procedure employs Carnoy's fixation followed by Protargol impregnation and Nissl staining. This technique permits the simultaneous observation of intracellular neurofibrils, neuronal processes and basophilic components of the neuron. In addition, it eliminates the need to stain alternate sections with separate procedures to view the various components of the urodele central nervous system.     

Silver Staining of Insect Central Nervous Systems by the Bodian Protargol Method

Improved fixation of ganglia of the central nervous system of Periplaneta americana and Schistocerca gregaria for silver staining by Power's (1943) modification of the Bodian protargol method is given by alcoholic Bouin aged for at least 40 days at 60° C. During impregnation of sections, increased copper and decreased pH give paler staining, more selective for nerve fibres. Prolonging impregnation from 24 to 48 hours weakens the stain and decreases selectivity. The intensity of the stain depends chiefly upon the amount of unreduced (developable) silver combined with the tissues; selectivity is determined mainly by the number and distribution of the reduced silver particles (‘nuclei’). In development, increased sodium sulphite gives more differentiation, increased hydroquinone gives less. Optimum developer composition depends upon impregnation, and thick sections need more differentiation than thinner ones. Within limits, change in one of the factors that control staining can be balanced by changes in others, but by suitable adjustment of the conditions the result can be varied from almost total staining of nerve fibres, for general neuroanatomy, to highly selective staining for tracing individual fibres.     

A Controllable Silver Stain for Nerve Fibers and Nerve Endings

A paraffin section method is described with a yellow-brown-black color range comparable to that of Ranson's pyridine silver block stain. After impregnation with activated protargol and reduction with a fine grain photographic developer, silver nitrate impregnation and reduction are repeated as often as necessary. The procedure is as follows:

Place hydrated sections of tissue fixed in chloral hydrate (25 g. in 100 ml. of 50% alcohol) in 1% aqueous protargol (Winthrop Chemical Co.) containing 5-6 g. metallic copper for 12-24 hours. After rinsing in 2 changes of distilled water, reduce 5 to 10 minutes in: Elon (Eastman Kodak Co.) 0.2 g., Na₂SO₃, dessicated, 10 g., hydroquinone 0.5 g., sodium borate powder 0.1 g., distilled water 100 ml. Wash thoroly in 4 or 5 changes of distilled water and place in 1% aqueous AgNO₃ for 10-20 minutes at 28°-50° C. Rinse in 2 or 3 changes of distilled water and reduce in the elon-hydroquinone solution. After thoroly washing in 4 or 5 changes of distilled water, examine under microscope.

If too pale, treat again in silver nitrate for 10-20 minutes, rinse, reduce 5-10 minutes and wash thoroly until nerve fibers show distinct microscopic differentiation, then dehydrate, clear and mount.     

Staining Paraffin Sections with Protargol 3. The Optimum pH for Reduction. 4. A Two-Hour Staining Method

Further work on conditions affecting the reduction of paraffin sections impregnated with protargol showed that the optimum pH for sulfite-amidol mixtures was between 6.5 and 7.5. A staining method which requires about two hours to complete consists of the following steps: (1) One hour impregnation at 60° C. in 10% AgNO₃. (2) Wash in distilled water 3 changes of 30 sec. each. (3) Put into protargol (Winthrop Chem. Co., New York, N. Y.) 0.2% aq. for another hour at room temperature. (4) Rinse 2 sec. (5) Reduce one to two min. in amidol 0.2 g., Na₂SO₃ 8 g., NaHSO₃ I g., and water 100 cc. (6) Wash thoroly. (7) Tone with 0.1% gold chloride. (8) Wash. (9) Reduce with a 0.5% aq. soln. of amidol (no sulfite). (10) Wash, dehydrate and cover. The method stains neurofibrillae and unmyelinated fibers and has worked well on most tissues of vertebrates. The stain follows acid alcoholic fixation.     

海洋纤毛虫--冠帆口虫的口器发生

对海洋盾纤目纤毛虫,冠帆口虫(Pleuronema coronatum)无性分裂期间口器的发生过程做了跟踪观察。结果显示其口器发生与已知的同属种普氏帆口虫具相似的过程。其口器发生及演化的基本模式可简述如下：老口区的三片小膜经历一个分化和移行过程;前仔虫的口侧膜来源于老口区的口侧膜;后仔虫的小膜1～3与口侧膜均来源于老的口侧膜;在口器发生过程中,前、后仔虫口侧膜的后端均形成一个相应的盾片区,其内的毛基粒在进入营养期之前被完全吸收[动物学报49(6)：829～834,2003]。     

A new soil hypotrich ciliate (Protozoa, Ciliophora) from Slovakia: Gonostomum albicarpathicum nov. spec

The morphology and infraciliature of the soil hypotrich ciliate, Gonostomum albicarpathicum nov. spec., collected from Biele Karpaty (White Carpathian Mts.), Slovakia, were investigated using living observations and the protargol impregnation method. This new species is distinguished from its congeners by a combination of characters including arrangement of macronuclear nodules forming a pair of nodules each ahead of and behind mid-body; 6-10 frontoventral cirri and 3 frontoterminal cirri forming a short row; first frontoterminal cirrus separated from the second by a slightly greater distance than between second and third.     

FISH probes for the detection of the parasitic dinoflagellate Amoebophrya sp. infecting the dinoflagellate Akashiwo sanguinea in Chesapeake Bay

A comparison of the small subunit rRNA sequences of a Chesapeake Bay strain of the dinoflagellate Akashiwo sanguinea and the dinoflagellate Amoebophrya sp. parasitizing it revealed several potential target sites that could be used to detect the parasite through in situ hybridization. The fluorescence of probed cells under various conditions of hybridization was measured by using a spot meter on a Nikon UFX-II camera attachment so that the effect of various hybridization parameters on probe binding could be determined. Probes directed against both the junction between helices 8 and 11 and helix 46 could detect the parasite, although the helix 8/11 probe produced a stronger signal under the conditions tested. The fluorescence of the probed cells increased with increasing hybridization time up to approximately twelve hours. The background fluorescence was lower at the wavelengths used to detect Texas Red than at those used to detect fluorescein, so probed cells were more distinct when Texas Red was used as the label. Cells stored in cold paraformaldehyde for a year still bound the probes. Young stages of the parasite could be seen more readily after in situ hybridization than after protargol impregnation.