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.     

Determination of the trophic state by qualitative and quantitative phytoplankton analysis in two gravel pit lakes

The phytoplankton of two gravel pits with comparable nutrient concentrations but different chlorophyll-a concentrations and phytoplankton biovolume was investigated qualitatively and quantitatively over a period of two years. This study deals with the phytoplankton-rich ‘Rotter See’ and the phytoplankton-poor ‘Paulsmaar’, both South of Cologne. The trophic state of both lakes was determined comparatively according to Ryding &; Rast (1989) and the categories used by Brettum (1989). The results were compared with each other. The trophic lake index according to Hörnström (1981) was modified slightly and subsequently determined for both lakes. A combination of qualitative and quantitative phytoplankton analysis made a proposal of a rehabilitation for the Rotter See possible.     

Contribution of ciliated protozoa to the planktonic biomass in a series of Ontario lakes: quantitative estimates and dynamical relationships

The plankton of nine Ontario lakes spanning several physiographicregions was sampled every two weeks during the ice-free periodof 1981, and one lake was studied in the three previous years.Phytoplankton, zooplankton, and ciliated protozoa were sampled,counted and sized. The size data were converted to biomass estimatesto yield quantitative comparisons of the relative allocationof biomass among different functional compartments. This isthe first study to look simultaneously and quantitatively atthe total plankton system of lakes (including ciliates, pbytoplanktonand net zooplankton) over a broad physiographic region. Ciliatesconstitute –10% of the non-algal biomass and 5% of thetotal planktonic biomass of these lakes. Ciliate standing cropsamong lakes are significantly corrrelated with total organicand total inorganic carbon concentrations in the water column,while the dynamics of ciliate biomass fluctuations are significantlycorrelated with variations in total phosphorus concentration,in conductivity, in Kjeldahl nitrogen concentration, and ininorganic carbon content. There appears to be a significantdynamical relationship between ciliates as a proportion of thetotal planktonic biomass, exclusive of filamentous and large(>30 µm) spherical algae, and the relative biomassof small algae (2–5 µm) as a fraction of total algalbiomass, again exclusive of filaments and large (>30 µm)algae. The hypothesis is advanced that ciliates primarily functionas bacterial grazers in planktonic ecosystems and that theirprimary competitors in this role are rotifers.     

Proficiencies of two artificial substrates in collecting stream macroinvertebrates

Proficiencies in collecting stream macroinvertebrates by two types of artificial substrates, #200 3M conservation webbing and cone-shaped concrete blocks, were compared with bottom net collections. Conservation webbing collected more individuals than cone substrates, but fewer taxa than either cone substrates or bottom nets. Diversity indices calculated for collections made with the three techniques were lowest for webbing collections, highest for net collections and intermediate for cone collections. It was concluded that collections made with webbing substrates gave an extremely distorted view of macroinvertebrate community balance due to a preponderance of net spinning and case building forms, and that cone substrates were better suited for use in the particular situation studied.Department of Biology, Virginia Polytechnic Institute and State University.Center for Environmental Studies, Virginia Polytechnic Institute and State University.     

The microfauna of algal mats and artificial substrates in Southern Victoria Land lakes of Antarctica

The first microfaunal colonization study of continental lakes in Antarctica is reported. In addition to the general taxonomic composition of six Antarctic lakes, detailed community composition analyses of two adjacent lakes, Lake Fryxell and Lake Hoare, using polyurethane foam artificial substrates suspended in the open water and placed directly over benthic algal mats are also presented. Protozoans, rotifers, tardigrades and nematodes constituted the microfaunal community in all lakes. Higher metazoans do not occur in lakes that we studied. Protozoans were the most diverse of the four groups, and the first dinoflagellates reported from southern Victoria Land, Gymnodinium and Glenodinium, were collected from Lake Fryxell. The greatest number of protozoan taxa (55) were associated with the algal mats in the more productive Lake Fryxell. Both the physical and chemical properties and taxonomic diversity showed marked variability between all lakes that were studied, especially the adjacent lakes, Fryxell and Hoare. Ecological variables which regulate taxonomic diversity in other lakes of the world also appear to exert similar influences in remote Antarctic lakes.     

Pelagic ciliated protozoa in two monomictic, southern temperate lakes of contrasting trophic state: seasonal distribution and abundance

Two lakes of contrasting trophic state in the central NorthIsland of New Zealand were sampled monthly for protozoan ciliatesand potential food resources. Oligotrichs dominated numbersin both lakes. Subdominants in oligotrophic Lake Taupo includedAskenasia, Pscudobalanion and Urotri-cha, and in eutrophic LakeOkaro Prorodon, Coleps, Urocentrum, Stentor and Spirostomumwere important. Biomass was dominated by large predatory ciliatesand Stentor in Lake Taupo, and Prorodon and Stentor in LakeOkaro. The importance of Prorodon and Stentor to ciliate biomassis unusual and has not been reported for northern hemispherelakes. Small ciliates (<20 µm) capable of consumingparticles <2 µm were a major component of the ciliatecommunity in Lake Taupo. Peaks in ciliate abundance occurredat the same time in both lakes: in autumn, at the beginningof mixis and in spring. Ciliates were vertically stratifiedduring mixis and stratification in both lakes. The effect wasmore pronounced during deoxygenation of the hypolimnion in LakeOkaro which excluded oligotrichs and introduced benthic ciliates.Ciliates were less abundant (mean 40001^–1 in Lake Okaroand 9001^–1 in Lake Taupo) than in comparable northerntemperate lakes. There was no correlation between the seasonaldistribution of ciliates and chlorophyll a, primarily causedby a winter peak in chlorophyll a dominated by large speciesof phytoplankton in Lake Taupo, at a time when ciliate numberswere low. The only consistent, significant correlations weretotal ciliate numbers and individual species of ciliates withbacterial concentrations in both lakes and with picophytoplanktonin Lake Taupo.     

Photon dependence of inorganic nitrogen transport by phytoplankton in perennially ice-covered antarctic lakes

¹⁵N was used to examine the influence of Photosynthetic Photon Flux Density (PPFD) on NO₃ ^– and NH₄ ⁺ transport by phytoplankton in the shallow and deep-chlorophyll layers of Lakes Fryxell and Vanda, Antarctica. The response observed in Lake Vanda could be modelled with a four parameter equation previously used to model photosynthesis. Only the 9 m NH₄ ⁺ transport experiments in Lake Fryxell could be modelled with this equation. Other experiments in Lake Fryxell showed either no response to PPFD or a linear increase with no saturation at the PPFD levels used. Distinct trends were observed in transport parameters both between depths and between nitrogen species. Overall, the parameters indicate that phytoplankton in these lakes possess nitrogen transport affinities similar to those reported for other aquatic systems.     

Chemical limnology of two artificial lakes used for both stormwater management and recreation

The aims of this study were to document the mainly chemical behaviour of two linked artificial lakes used for both stormwater management and recreation in the new town of Craigavon. Further, the understanding of their behaviour should help in their management and the design of other similar lakes.The lake mean total phosphorus (73 µg P l^–1), nitrate (0.50 mg N l^–1) and chlorophyll a (25 µg l^–1) concentrations, Secchi depth (1.2 m) and the estimated total phosphorus loading (1.98 g m^–2 a^–1) all classify the main lake as eutrophic. An important source of the phosphorus load on the lakes is the urban area of Craigavon (52% of the total load). The interrelationships between total phosphorus, chlorophyll a and Secchi depth in the main lake are similar to those in natural ones. In addition, the lake follows the total phosphorus load — trophic state relationships (lake total phosphorus and chlorophyll a concentrations and Secchi depth) found to apply elsewhere. These two points indicate that the artificial lakes in Craigavon behave similarly to natural ones.     

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.     

Methods of denaturation and renaturation of DNA in interphasic chromatin: cytochemical quantitative analysis by Methyl Green staining

Almost diploid nuclei (as judged from the microdensitometric evaluation of the Feulgen positive material) of granular and Purkinje cells of the rat cerebellar cortex, were submitted to in situ DNA denaturation and renaturation experiments. We assessed the double-strandedness of DNA, by Methyl Green staining according to Scott (1967). Under these conditions a stoichiometric ratio between bound dye and DNA exists, suitable for quantitative microdensitometric measurements. Our data show that DNA in the interphasic chromatin is never completely denatured after the treatments we used. Furthermore, the renaturation takes place in a different way in the two cell types. Owing to the unlike chromatin packing of granular and Purkinje nuclei, we suggest that nuclear proteins must interfere differently on the in situ denaturation and renaturation processes.     

Semi-automated recognition of protozoa by image analysis

A programme was created to semi-automatically analyse protozoal digitised images. Principal Component Analysis technique was used for species identification. After data collection and mathematical treatment, a three-dimensional representation was generated and several protozoa (Opercularia, Colpidium, Tetrahymena, Prorodon, Glaucoma and Trachelophyllum) species could be positively identified.     

露天采矿形成的人工湖中的水鸟建群(英文)

1999-2003年,在捷克共和国特布盆地生物圈保护区(Tebo Basin Biosphere Reserve)内,我们对13个采矿后形成的人工湖中水鸟群落的形成过程进行了研究.我们假定生活在邻近鱼塘中的水鸟会逐渐向这些人工湖迁移和聚集.在这些人工湖中共记录到42种水鸟,隶属于10个目,其中17种典型的海滨鸟类是该群落的重要组成部分.这些水鸟的季节动态过程与各物种自身的生活史阶段有关(如筑巢、迁徙),同时也被人类的采矿活动所影响.环境因素和人类干扰影响了绿头鸭(Anas platyrhynchos)、疣鼻天鹅(Cygnus olor)和凤头鸊鷉(Podiceps cristatus) 等优势种的分布.沙坑湖作为一种独特的生境为本保护区的濒危物种提供了栖息地,如:小苇(开鸟)(Ixobrychus minutus)、大苇莺(Acrocephalus arundinaceus)及其它潜在分布的鸟类.对南波西米亚地区沙坑湖鸟类的研究,可为其它相似地区水鸟群落演替研究提供了重要经验     

Streptavidin-based quantitative staining of intracellular antigens for flow cytometric analysis.

A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 micrograms/ml to 5 micrograms/ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed.     

Silver staining in pinealocyte nuclei: Observations and quantitative analysis during the rat oestrous cycle

Summary Morphology, distribution and number of different argyrophilic aggregates appearing in pinealocyte nuclei have been studied during the rat oestrous cycle. Using the Ag-NOR reaction we have found two types of argyrophilic aggregates in pinealocyte nuclei. One of them, corresponding to the fibrillar centres and the surrounding fibrillar component, appears in the nucleoli. The silver grains are more loosely packed in the dense fibrillar component than in the fibrillar centres. A decrease in the number of these nucleolar argyrophilic aggregates was obtained at oestrous. A second type of silver grain aggregate was observed in the pinealocyte nucleoplasm. We call them Ag-granule clusters because they are similar to the interchromatin granule clusters and constitute the only silver deposit forming aggregates apart from the nucleolus. The number of Ag-granule clusters is significantly smaller at oestrous and meta-oestrous than at dioestrous and pro-oestrous.     

Ciliated protozoa and water quality in the Parma River (Northern Italy): long-term changes in the community structure

Ciliated protozoans from sediments of the River Parma (Northern Italy) were collected from 7 stations, selected according to their hydrological, chemical, and physical characteristics, and to river zonation during autumn 1989. Fiftythree species were observed. A saprobic evaluation of the river was made using the taxocenoses observed, and the results were compared with data collected in 1978, when the River Parma still received untreated sewage. In 1989 the hilly section of the stream was found to be more polluted than in 1978, but in the plain section, the saprobic indices were now slightly lower. The marked change in the ciliate community of the hills was accompanied by an increase in organic load, which caused the disappearance of some typical species and dampened the diversifying effects of other environmental factors, producing comparatively similar community structures in that whole zone.     

Colonization of artificial substrates by stream insects: Influence of substrate size and diversity

A field experiment was performed in a New Mexico (U.S.A.) stream to investigate the relation between size and diversity of substrate and numbers and diversity of colonizing insects. Baskets with either small gravel, large gravel, or a 1 : 1 mixture of the two sizes were placed in a riffle area and colonization was monitored for 19 days.Colonization was rapid, and both total number of individuals and number of species colonizing the substrates had stopped increasing by the end of the experiment. Fewer individuals colonized baskets downstream, suggesting that the drift is a major source of insect colonists. Small substrate supported more individuals and more species than the larger stones. Total number of individuals and the number of species on the mixed substrate were between numbers on small and large substrate. These results do not support previous generalizations on the relation between substrate size and complexity and the structure of invertebrate communities in streams. Results of the study are compared with the findings of recent experimental studies and it is suggested that earlier generalizations on the role of substrate size and complexity need to be reexamined.Order of authors was determined by the flip of a coin. Reprint requests may be sent to either author.     

Application of discriminant analysis and quantitative cytologic examination to gastric lesions

OBJECTIVE: To investigate of the potential value of morphometry and discriminant analysis for the classification of benign and malignant gastric cells and lesions. STUDY DESIGN: The data set consisted of 13,300 cells from 120 cases composed of 30 cases of cancer, 26 cases of gastritis and 64 cases of ulcer according to the final histologic diagnosis. The cytologic diagnosis was divided into 5 categories (gastritis, ulcer, inflammatory dysplasia, cancer and true dysplasia). Classification was attempted at 2 levels: the cell level to classify individual cells and the case level to classify individual cases. For the cellular classification the measured cells from 50% of available cases were selected as a training set to construct a model. The cells from the remaining cases were used as a test set to validate the model. Similarly for case classification, the same 50% of cases that were used for cell classification were used as a training set and the remaining cases as a test set. Images of routinely processed gastric smears stained by the Papanicolaou technique were analyzed by a customized image analysis system. RESULTS: Application of discriminant analysis on the test set gave correct classification of 98.4% of benign cells and 67.1% of malignant cells. On case classification, 100% accuracy was achieved for benign and malignant cases, both for the training and test sets. CONCLUSION: The application of discriminant analysis described in this paper could produce significant classification results at the cellular and individual case level.     

Phytoplankton community dynamics of two adjacent Dutch lakes in response to seasons and eutrophication control unravelled by non-supervised artificial neural networks

Long-term time-series of the eutrophic Dutch lakes Veluwemeer and Wolderwijd were subject to ordination and clustering by means of non-supervised artificial neural networks (ANN). A combination of bottom-up and top-down eutrophication control measures has been implemented in both lakes since 1979. Dividing time-series data from 1976 to 1993 into three distinctive management periods has facilitated a comparative analysis of the two lakes regarding both the seasonal and long-term dynamics in response to eutrophication control. Results of the study have demonstrated that non-supervised ANN are an alternative technique: (1) to elucidate causal relationships of complex ecological processes, and (2) to reveal long-term behaviours of ecosystems in response to different management approaches. It has been shown that external nutrient control combined with food web manipulation have turned both lakes from nitrogen to phosphorus limitation, and from blue-green algae to diatom and green algae dominance.     

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.     

Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining

Fibers in cross sections of human and rat muscle were typed by using histochemical ATPase stains, and the results were compared with those of quantitative enzyme assays of fragments of the same fibers dissected from serial freeze-dried sections. Two enzymes previously used to assess the metabolic type were measured in each case: lactate dehydrogenase and either adenylokinase (human fibers) or malate dehydrogenase (rat fibers). With human fibers there was essentially complete agreement between ATPase staining and the metabolic enzyme assays in distinguishing types I and II fibers. The agreement was less consistent with regard to type IIA and IIB fibers. A number of ATPase type IIC fibers were identified in one human muscle, and were found to fall between ATPase types I and IIA on the basis of metabolic enzyme assay results. Rat-fiber ATPase types I, IIA, and IIB from the plantaris muscle were rather well segregated on a two-dimensional lactate dehydrogenase-malate dehydrogenase grid. In the rat soleus muscle, ATPase types I and IIA fibers were shifted to lower lactate dehydrogenase levels, with IIC fibers interposed between them.