Aerobic cultivation of anaerobic rumen protozoa, Entodinium caudatum and Epidinium caudatum.

Rumen protozoa, primarily ciliates, are one of the important groups of strictly anaerobic microbes living in the rumen. Despite their ubiquitous occurrence in the rumen and significant contribution to host animals, it is still poorly understood why they live only in the rumen and similar environment. Because rumen protozoa require strict anaerobic conditions to sustain their viability and grow, only a few laboratories equipped with protozoology expertise and anaerobic facilities can grow rumen protozoa in laboratory. Also for the same reason, only a few species have been grown and maintained as laboratory cultures for research. Prompted by a recent study, we hypothesized that anaerobic rumen protozoa could also be cultivated aerobically if antioxidants were included in the media. Indeed, our experiments showed that the cultures of both Entodinium caudatum and Epidinium caudatum, two major rumen protozoal species, could be cultured successfully in aerobic media supplemented with ascorbic acid, glutathione and α-ketoglutarate as antioxidants. Anaerobic fermentation was maintained through the fermentation characteristics and microbial populations were altered to some extent under aerobic conditions. The antioxidants also enhanced the revival of cryopreserved stock cultures of both rumen protozoal species. The results of this study may facilitate and promote future research in which rumen protozoa need to be cultured in laboratory.

Can fungal zoospores be the source of energy for the rumen protozoa Eudiplodinium maggii?

Results of our earlier studies showed the ability of ciliates Eudiplodinium maggii to digest and metabolize commercial chitin. The natural source of this polysaccharide in the rumen are fungi. The objectives of present research were to determine the effect of fungal zoospores on the survival and population density of E. maggii to quantify the concentration of chitin in the cells of protozoa and to examine the ability of E. maggii, to ferment chitin of fungal zoospores. The cultivation experiment showed that the survival of protozoa was shorter than 4 days when the culture medium was composed of buffer solution and lyophilized fungal spores. An enrichment of this medium with wheat gluten prolonged the survival of ciliates up to 8 days. The supplementation of the last medium with meadow hay enabled the protozoa to survive for 28 days but a positive effect was observed only during the last 8 days of experiment. The chitin content was 0.27 ng and 0.21-0.35 ng per single zoospore and ciliate, respectively. An increase in the concentration of volatile fatty acids (VFA) was found when protozoa were incubated with zoospores. The production rate of VFA was 46.3 pM/protozoan per h whereas the endogenous production did not exceed 31 pM/protozoan per h. The molar proportion of acetic acid was 77.7% and these of butyric and propionic acids-12.2 and 11.0%, respectively. The obtained results make it evident that carbohydrates present in fungal zoospores were utilized by protozoa in energy yielding processes.

Rumen fermentation and microbial population in lactating dairy cows receiving diets containing oilseeds rich in C-18 fatty acids.

Sixteen Holstein rumen-cannulated primiparous milking dairy cows were fed a control diet (CN) based on maize silage and soyabean meal during a 4-week period before the start of a 21-d experiment with oilseeds containing high concentration of linoleic acid (Linola™) or linolenic acid (NuLin™). Thereafter, four cows received ad libitum one of each of four dietary treatments comprising of CN, Linola (LN), NuLin (NL) and LN/NL (50/50 % combination). Each LN, NL and LN/NL treatment contained 6 % oil of DM. Rumen digesta samples were collected on days 6, 11, 16 and 21 and milk samples on days 13, 15 and 17. There were no effects (P>0.05) of the oilseeds on pH and concentrations of NH3-N and total volatile fatty acids, while the acetate:propionate ratio was decreased (P< 0.05). The oilseeds also decreased (P< 0.05) protozoa and increased (P< 0.1) total cellulolytic bacteria in rumen fluid, especially when containing high dietary linoleic acid (P< 0.05). The milk protein concentration was increased (P< 0.1) by the dietary linoleic acid, which produced most beneficial results. It was concluded that supplements of linoleic acid in diets of ruminants might contribute to better digestion of dietary fibre and increased quality of milk.

Re-alimentation strategy to manoeuvre body condition and carcass characteristics in cull ewes.

Improvement in body condition and carcass traits through nutritional intervention was studied in cull ewes. Sixty-eight adult non-productive Malpura ewes (average body weight 26.7 ± 0.33 kg) were randomly divided into four equal groups: G0 maintained on free grazing for 8 h on protected natural rangeland with ad libitum guar (Cyamopsis tetragonoloba) straw (GS) after grazing; G1, G2 and G3 fed with supplemental concentrate at the rate of 250 g, 2.5% of live weight (LW) and ad libitum, respectively. The experiment was continued for 90 days and daily feed intake, weekly LW and body condition score (BCS) were recorded. Intake and digestibility of nutrients were assessed by indicator method. Rumen fermentation attributes and blood biochemical profile were studied to assess the dietary effects and animals were slaughtered at the end of experiment for evaluation of carcass characteristics. Higher dry matter (DM) intake and improvement in plane of nutrition was observed in G2 and G3 with a higher LW gain (LWG) and improvement in BCS than in G0. The digestibility of DM, organic matter, CP, ADF and cellulose was higher (P < 0.05) in G2 and G3 than in G0. A lower ruminal pH and ammonia N but higher total N and trichloroacetic acid-precipitable N, an increase in holotrichs, spirotrichs and total protozoa population, increase in haemoglobin but decrease in serum total cholesterol and non-esterified fatty acids were observed in high-concentrate-fed groups. Carcass attributes revealed increase (P < 0.05) in empty LW, dressing yield, eye muscle area, subcutaneous and intramuscular fat, decrease in shear force value and higher (P < 0.05) protein content in Longissimus dorsi muscle in test groups than in the control. Above all, the G2 animals had better rumen environment and blood biochemical attributes and consumed more feed with enhanced digestibility that supported higher LWG at better feed conversion efficiency, improvement in BCS and carcass quality. Thus, re-alimentation of cull ewes with challenged feeding of concentrate at 2.5% of LW on a basal roughage diet for a period of 3 months may have promise for better economic return to the farmers with possibly meeting quality mutton for human consumption.

[Ciliate diversity and spatiotemporal variation in surface sediments of Yangtze River estuary hypoxic zone].

By using denaturing gradient gel electrophoresis (DGGE) and sequencing as well as Ludox-QPS method, an investigation was made on the ciliate diversity and its spatiotemporal variation in the surface sediments at three sites of Yangtze River estuary hypoxic zone in April and August 2011. The ANOSIM analysis indicated that the ciliate diversity had significant difference among the sites (R = 0.896, P = 0.0001), but less difference among seasons (R = 0.043, P = 0.207). The sequencing of 18S rDNA DGGE bands revealed that the most predominant groups were planktonic Choreotrichia and Oligotrichia. The detection by Ludox-QPS method showed that the species number and abundance of active ciliates were maintained at a higher level, and increased by 2-5 times in summer, as compared with those in spring. Both the Ludox-QPS method and the DGGE technique detected that the ciliate diversity at the three sites had the similar variation trend, and the Ludox-QPS method detected that there was a significant variation in the ciliate species number and abundance between different seasons. The species number detected by Ludox-QPS method was higher than that detected by DGGE bands. Our study indicated that the ciliates in Yangtze River estuary hypoxic zone had higher diversity and abundance, with the potential to supply food for the polyps of jellyfish.

Analysis of expressed sequence tags (ESTs) and gene expression changes under different growth conditions for the ciliate Anophryoides haemophila, the causative agent of bumper car disease in the American lobster (Homarus americanus).

The scuticociliate Anophryoides haemophila, causes bumper car disease in American lobster (Homarus americanus) in commercial holding facilities in Atlantic Canada. While the parasite has been recognized since the 1970s and much has been learned about its biology, minimal molecular characterization exists. With genome consortiums turning to model organisms like the ciliates Tetrahymena and Paramecium, the amount of relevant sequence data available has made sequence surveys more attractive for gene discovery in related ciliates. We sequenced 9984 expressed sequence tags (ESTs) from a non-normalized A. haemophila cDNA library to characterize gene expression patterns, functional gene distribution and to discover novel genes related to the parasitic life history. The A. haemophila ESTs were grouped into 843 clusters and singletons with 658 EST clusters having identifiable homologs, while 159 ESTs were unique and had no similarity to any sequences in the public databases. Not unexpectedly, about 67% of the A. haemophila ESTs have similarity to annotated and hypothetical genes from the related oligohymenophorean ciliate, Tetrahymena. Numerous cysteine proteases, hypothetical proteins and novel sequences possess putative secretory signal peptides suggesting that they may contribute to the pathogenesis of bumper car disease in lobster. Real time RT-qPCR analysis of cathepsin L and two homologs of cathepsin B did not show any changes in gene expression under varying in vitro growth conditions or during a modified-in vivo infection which may be suggestive of the opportunistic life history strategy of this ciliate.

The effect of anoxia and anaerobia on ciliate community in biological nutrient removal systems using laboratory-scale sequencing batch reactors (SBRs).

Little is known about the effect of anaerobic and anoxic stages on the protozoan community in the activated sludge process and how this subsequently affects performance. Using a laboratory-scale BNR system the effect of different periods of anoxia on both the protozoan community and performance efficiency have been examined. Four SBRs were operated at two cycles per day using a range of combined anoxic/anaerobic periods (0, 60, 120 and 200 min). Effluent quality (TOC, BOD, TP, TN, NH(4)-N, NO(3)-N and NO(2)-N), sludge settleability and ciliate community (species diversity and abundance) were analysed over a periods of up to 24 days of operation. The species richness and total abundance of ciliates were found to decrease with longer anoxic/anaerobic periods. Both, positive and negative significant correlations between the abundance of certain species and the period of anoxia was observed (e.g. Opercularia microdiscum, Epicarchesium granulatum), although other species (i.e. Acineria uncinata, Epistylis sp.) were unaffected by exposure to anoxia. In the laboratory-scale units, the 60 min anoxic/anaerobic period resulted in good process performance (TOC and BOD removal of 97-98% respectively), nitrification (80-90%), denitrification (52%) but poor levels of biological P-removal (12%); with the protozoan community moderately affected but still diverse with high abundances. Increasing the length of anoxia to up to 200 min did not enhance denitrification although P-removal rates increased to between 22 and 33%; however, ciliate species richness and total abundance both decreased and sludge settleability became poorer. The study shows that activated sludge ciliate protozoa display a range of tolerances to anoxia that result in altered ciliate communities depending on the length of combined anoxic/anaerobic periods within the treatment process. It is recommended that anoxic/anaerobic periods should be optimised to sustain the protozoan community while achieving maximum performance and nutrient removal.

A mesocosm study of the changes in marine flagellate and ciliate communities in a crude oil bioremediation trial.

Protozoan grazers play an important role in controlling the density of crude-oil degrading marine communities as has been evidenced in a number of microcosm experiments. However, small bioreactors contain a low initial titre of protozoa and the growth of hydrocarbon-depleting bacteria is accompanied by the fast depletion of mineral nutrients and oxygen, which makes microcosms rather unsuitable for simulating the sequence of events after the oil spill in natural seawater environment. In the present study, the population dynamics of marine protozoan community have been analysed in a 500 l mesocosm experiment involving bioaugmented oil booms that contained oil sorbents and slow-release fertilisers. A significant increase in numbers of marine flagellates and ciliates on biofilms of oil-degrading microbes was microscopically observed as early as 8 days after the start of the experiment, when protozoa exhibited a population density peak making up to 3,000 cells ml(-1). Further, the protozoan density varied throughout the experiment, but never dropped below 80 cells ml(-1). An 18S rRNA gene-based fingerprinting analysis revealed several changes within the eukaryotic community over the whole course of the experiment. Initial growth of flagellates and small ciliates was followed by a predominance of larger protozoa. According to microscopic observations and SSU rRNA molecular analyses, most predominant were the ciliates belonging to Euplotidae and Scuticociliatia. This is the first study to characterise the eukaryotic communities specifically in a large-scale oil bioremediation trial using both microscopy-based and several molecular techniques.

Relationship of protozoan biomass to phosphate and nitrate removal from activated sludge mixed liquor.

The relationship between protozoan biomass concentration and phosphate and nitrate removal was investigated in mixed liquor using three different carbon sources as supplements. The study was carried out using three respective initial biomass concentrations in a shaking flask environment. Samples were taken every 24 h to determine phosphate, nitrate, dissolved oxygen and chemical oxygen demand. The results revealed a direct relationship between decreases in nutrient concentrations and increases in cell densities of the isolates. Between 24 and 96 h, the increases in the protozoan density corresponded to a phosphate decreases from initial ranges of 55.42-57.36 mg/L, 50.27-51.17 mg/L and 50.01-50.83 mg/L to final ranges of 2.46-11.90 mg/L, 0.61-11.80 mg/L and 1.29-13.89 mg/L, in the presence of Aspidisca, Trachelophyllum and Peranema, respectively. Nitrate concentrations were observed to decrease from initial ranges of 23.84-25.90 mg/L, 23.94-25.84 mg/L and 26.12-26.54 mg/L to final ranges of 0.11-6.32 mg/L, 0.16-5.60 mg/L and 0.24-9.04 mg/L, respectively. The study had revealed that an increase in cell density of the test isolates produces a corresponding increase in phosphate and nitrate removal.

Does microorganism stoichiometry predict microbial food web interactions after a phosphorus pulse?

Knowledge of variations in microbial food web interactions resulting from atmospheric nutrient loads is crucial to improve our understanding of aquatic food web structure in pristine ecosystems. Three experiments mimicking atmospheric inputs at different nitrogen/phosphorus (N/P) ratios were performed in situ covering the seasonal biological succession of the pelagic zone in a high-mountain Spanish lake. In all experiments, abundance, biomass, algal cell biovolume, P-incorporation rates, P-cell quota, and N/P ratio of algae strongly responded to P-enrichment, whereas heterotrophic bacteria remained relatively unchanged. Ciliates were severely restricted when a strong algal exploitation of the available P (bloom growth or storage strategies) led to transient (mid-ice-free experiment) or chronic (late ice-free experiment) P-deficiencies in bacteria. In contrast, maximum development of ciliates was reached when bacteria remained P-rich (N/P < 20) and algae approached Redfield proportions (N/P approximately 16). Evidence of a higher P-incorporation rate supports the proposition that algae and bacteria shifted from a mainly commensalistic-mutualistic to a competitive relationship for the available P when bacterial P-deficiency increased, as reflected by their unbalanced N/P ratio (N/P > 20-24). Hence, the bacterial N/P ratio proved be a key factor to understand the algae-bacteria relationship and microbial food web development. This study not only demonstrates the interdependence of life history strategies, stoichiometric nutrient content, and growth but also supports the use of bacterial N/P thresholds for diagnosing ciliate development, a little-studied aspect worthy of further attention.

Nature of phosphorus limitation in the ultraoligotrophic eastern Mediterranean.

Phosphate addition to surface waters of the ultraoligotrophic, phosphorus-starved eastern Mediterranean in a Lagrangian experiment caused unexpected ecosystem responses. The system exhibited a decline in chlorophyll and an increase in bacterial production and copepod egg abundance. Although nitrogen and phosphorus colimitation hindered phytoplankton growth, phosphorous may have been transferred through the microbial food web to copepods via two, not mutually exclusive, pathways: (i) bypass of the phytoplankton compartment by phosphorus uptake in heterotrophic bacteria and (ii) tunnelling, whereby phosphate luxury consumption rapidly shifts the stoichiometric composition of copepod prey. Copepods may thus be coupled to lower trophic levels through interactions not usually considered.

Trophic interactions within the microbial food web in a tropical floodplain lake (Laguna Bufeos, Bolivia)

Whether the primary role of bacterioplankton is to act as "remineralizers" of nutrients or as direct nutritional source for higher trophic levels will depend on factors controlling their production and abundance. In tropical lakes, low nutrient concentration is probably the main factor limiting bacterial growth, while grazing by microzooplankton is generally assumed to be the main loss factor for bacteria. Bottom-up and top-down regulation of microbial abundance was studied in six nutrient limitation and dilution gradient-size fractionation in situ experiments. Bacteria, heterotrophic nanoflagellates (HNF), ciliates and rotifers showed relatively low densities. Predation losses of HNF and ciliates accounted for a major part of their daily production, suggesting a top-down regulation of protistan populations by rotifers. Phosphorus was found to be strongly limiting for bacterial growth, whereas no response to enrichment with Nitrogen or DOC was detected. HNF were the major grazers on bacteria (g=0.43 d-1 ), the grazing coefficient increased when ciliates were added (g=0.80 d-1 ) but decreased when rotifers were added (g=0.23 d-1 ) probably due to nutrient recycling or top-down control of HNF and ciliates by rotifers

Que el bacterioplancton juegue básicamente un papel de reciclaje de nutrientes, o sea una fuente directa de nutrientes, depende de varios factores que afectan su producción y abundancia. En los lagos tropicales, la baja concentración de nutrientes es posiblemente el principal factor limitante del crecimiento bacteriano, y suele suponerse que la mayor pérdida poblacional de bacterias se debe a depredación por parte del microzooplancton. Estudiamos la regulación ascendente ("de abajo hacia arriba") y descendente ("de arriba hacia abajo") de abundancia bacteriana mediante seis experimentos in situ de limitación de nutrientes y de fraccionamiento de la dilución tamaño- gradiente. Bacterias, nanoflagelados heterotróficos (NHT), ciliados y rotíferos tienen densidades relativamente bajas. Las pérdidas por depredación de ciliados y de NHT explicaron la mayoría de la producción diaria, lo que sugiere que las poblaciones de protistas son reguladas por los rotíferos de forma descendente. El fósforo resultó ser un limitante fuerte del crecimiento bacteriano, pero no se detectó efecto del enriquecimiento con nitrógeno ni con "DOC". Los NHT fueron los principales depredadores de bacterias (g=0.43 d-1 ). El coeficiente de ramoneo aumentó al agregar los ciliados (g=0.80 d-1 ) pero disminuyó al agregar los rotíferos (g=0.23 d-1 ) probablemente debido a reciclaje de nutrientes o a control descendente de los NHT y ciliados por parte de los rotíferos

Trophic interactions within the microbial food web in a tropical floodplain lake (Laguna Bufeos, Bolivia).

Whether the primary role of bacterioplankton is to act as "remineralizers" of nutrients or as direct nutritional source for higher trophic levels will depend on factors controlling their production and abundance. In tropical lakes, low nutrient concentration is probably the main factor limiting bacterial growth, while grazing by microzooplankton is generally assumed to be the main loss factor for bacteria. Bottom-up and top-down regulation of microbial abundance was studied in six nutrient limitation and dilution gradient-size fractionation in situ experiments. Bacteria, heterotrophic nanoflagellates (HNF), ciliates and rotifers showed relatively low densities. Predation losses of HNF and ciliates accounted for a major part of their daily production, suggesting a top-down regulation of protistan populations by rotifers. Phosphorus was found to be strongly limiting for bacterial growth, whereas no response to enrichment with Nitrogen or DOC was detected. HNF were the major grazers on bacteria (g-0.43 d(-1)), the grazing coefficient increased when ciliates were added (g- 0.80 d(-1)) but decreased when rotifers were added (g- 0.23 d(-1)) probably due to nutrient recycling or top-down control of HNF and ciliates by rotifers.

The effects of organic selenium supplementation on the rumen ciliate population in sheep.

The effect of selenium supplementation on the rumen protozoan population of sheep was demonstrated. Both the total and generic counts of rumen ciliates in sheep fed a diet with basal Se content (70 microg/kg dry matter) were compared to those of animals given feed supplemented with inorganic (disodium selenite) or organic Se (selenized yeast) (310 microg/kg dry matter). The genera of Entodinium, Isotricha, Dasytricha, Ophryoscolex, Diploplastron and Polyplastron occurred in all sheep except for the control, in which Ophryoscolex was not observed. The population of Ophryoscolex caudatus f. tricoronatus was significantly higher in sheep supplemented with organic Se than in animals given inorganic Se (by 160 %). Supplementation of feed with selenized yeast induced significant growth in the Diploplastron population (by 63 %) while no change occurred in sheep given selenite. The populations of Dasytricha ruminantium and Polyplastron multivesiculatum were higher than control in both Se-supplemented groups. The ciliate population of Entodinium spp. was not influenced by Se supplements. Our results suggest a protective effect of Se feed supplementation on the development of some rumen ciliate species in young ruminants.

In vitro growth requirements for the fish pathogen Philasterides dicentrarchi (Ciliophora, Scuticociliatida).

Philasterides dicentrarchi is a scuticociliate causing fatal disease in farmed turbot and sea bass. In view of its high virulence and endoparasitic location, this parasite cannot be effectively controlled by formalin baths, and no systemic chemotherapeutic treatments have yet proved effective; immunoprophylaxis may thus be an attractive alternative approach. Since vaccine development is greatly facilitated by axenic culture of the pathogen, we have developed a simple axenic culture system based on commercially available Leibovitz L-15 medium, supplemented with fetal bovine serum, lipids (lecithin and Tween 80), nucleosides and glucose. After 1 week's culture under optimal conditions (salinity 10 per thousand, pH 7.2, temperature between 18 and 23 degrees C), yields of 1-2 x 10(5)cells/ml were obtained. Even cultures with seeding densities as low as 20 cells/ml were found to produce a good yield of ciliates (about 6 x 10(4)cells/ml) after 11 days of incubation. The ciliates thus obtained were free of contamination by other microorganisms, enabling preparation of pure P. dicentrarchi antigens for vaccine development studies.

Cell type-specific gene expression in the cell cycle of the dimorphic ciliate Eufolliculina uhligi.

The life cycle of the unicellular eukaryote Eufolliculina uhligi includes two structurally and physiologically different cell types: a motile swarmer that is arrested in the cell cycle, and a sessile cell (trophont) that feeds and reproduces. These two cell types offer an exceptionally favourable system for the isolation of genes involved in cell cycle regulation and cellular morphogenesis. Differential screening of a trophont cDNA library using a swarmer-subtracted, trophont-specific probe yielded eleven clones that represent trophont-specific transcripts and one clone that represents a swarmer-specific transcript. Sequence analysis showed that seven clones, including the only swarmer-specific one, represent unknown genes, whereas five clones could be identified by sequence comparisons. Two of the clones appear to encode proteins that are involved in the regulation of growth and metabolism. The deduced sequences of three clones resemble potential cell cycle regulators. Data are presented on a putative member of the calcium/calmodulin-dependent protein kinase family and on a TIP120-like sequence, which is the first such sequence to be described since the discovery of the rat TIP120 protein. Furthermore, a unique new sequence is presented, whose features suggest that it represents a protein that is involved in the regulation of cell division. It includes domains characteristic of two different protein families, cyclin-dependent kinases (CDKs) and cyclins, both of which are known to be cell cycle regulators. Based on our results we propose a model for cell cycle regulation in ciliated protozoa.

Overview of "bumper car" disease--impact on the North American lobster fishery.

Recent (1993) landings of American lobsters (Homarus americanus) were valued at $294 million (Can.) in Canada and $213 million (Can.) in the United States. However, post-harvest losses are estimated at $50-75 million (10-15%) annually. The lobster fishery is one of the few remaining viable traditional fisheries in eastern North America. "Bumper car" disease of lobsters, caused by the scuticociliate Anophryoides haemophila, can cause significant losses in coldwater impoundments. Apparently epidemics now occur more frequently and with greater severity; surprisingly the epidemiology and economic impacts of "bumper car" disease are not well documented. The ciliate A. haemophila is easily maintained in a cell-free, chemically defined, seawater-based medium at 5 degrees C. Cultured ciliates require longer and more parasites to kill lobsters than those transmitted by intrahaemocoelic injection from lobster-to-lobster. Regardless of source of ciliates, the larger the inoculum, the more rapid the death of lobsters. The pathogenesis of "bumper car" disease is unknown. Horizontal transmission could occur across the thin cuticle of gills or via wounds in the exoskeleton present during moulting of lobsters. Because ciliates are initially sequestered in lobster tissues for an extended period, they are detectable sooner by histological examination of tissues than by direct examination or culture of haemolymph. Additional to indirect fluorescent antibody testing and immunoperoxidase staining of tissues, utilizing monoclonal antibodies prepared to sonicated ciliates, the parasites are readily detected with oligonucleotide probes based on ssu-rDNA of A. haemophila. The prevalence of A. haemophila should be re-evaluated. Ciliates sequester in gill, heart and muscle tissues. Several disinfectants and chemotherapeutants, licensed in North America for veterinary use in food-producing animals, are efficacious against A. haemophila in vitro. A definition of healthy vs ciliate-infected lobsters is being prepared, based on haematology and clinical chemistry of haemolymph. Our novel bar-coded labelling system for aquatic organisms facilitates experimental design and randomization protocols of lobsters. The model of "bumper car" disease will aid study of health and infectious disease processes of lobsters and other crustaceans.

[Synthesis of RNA and DNA in isolated nuclei of Colpoda cucullus (Protozoi Ciliati)]. / Sintesi di RNA e DNA nei nuclei isolati di Colpoda cucullus (Protozoi Ciliati).

The ciliated protozoan Colpoda cucullus has been cultivated at 27 degrees C with gentle shaking in a baked lettuce infusion supplemented with Klebsiella suspensions. Under these conditions cells had a mean generation time of about 7 hours and could attain densities up to 20,000/ml and 45,000/ml in the log and stationary phase of growth, respectively. Nuclear preparations obtained from exponentially growing cells by the gum arabic-octanol method showed a satisfactory degree of purity and integrity. They consisted primarily of the large macronuclei attached to which the small micronuclei were sometimes visible. Upon incubation at 27 degrees C in conventional reaction mixtures nuclear preparations actively incorporated 3H-UTP and 3H-dTTP into acid-insoluble material. alpha-amanitin caused a 50% inhibition of RNA synthesis whereas aphidicolin did not affect at all DNA synthesis.