Isolation and analysis of particulate detritus as an aid to studies on the nutritional ecology of deposit feeders

By submitting detrituss.l., consisting of the fractions particulate detrituss.s., mineral particles, mesofaunal elements, cyanobacterial colonies and other microalgae such as diatoms, to density gradient centrifugation (DGC) in Percoll and Ludox-TM the major part of detrituss.s. could be separated. Qualitative analysis of the fractions proved that the organic carbon content and (to a small extent) the nitrogen content of the fractions distinguished after DGC were higher than those of the detrituss.l. before DGC, probably due to contamination with the gradient material. These results as well as the problems of quantifying the natural food of deposit feeders are discussed.     

Aspects of nitrogen fixation in Lough Neagh. II. Competition between Aphanizomenon flos-aquae, Oscillatoria redekei and Oscillatoria agardhii

SUMMARY.

• 1 The effect of nutrient availability on the growth of natural samples of Lough Neagh plankton (Aphanizomenon flos-aquae, initial innoculum 0.98 mm³ l^-1, Oscillatoria redekei, 2.52 mm³ l^-1 and Oscillatoria agardhii, 4.96 mm³ l^-1) was studied, using ecologically realistic conditions of temperature (15°C), nitrate-N (<0.35g m^-3) and orthophosphate-P (<0.55g m^-3), in which the combinations –N–P, –N+P, +N–P, and +N+P were used.
• 2 After 8 days A. flos-aquae had become dominant in treatments lacking N. After 12 days with N available the mean yields of O. redekei (37.7 mm³ l^-1) and O. agardhii (10.82 mm³ l^-1) were significantly greater (P=5%) than their respective yields (3.6 and 2.6 mm³ l^-1) in the absence of N. Somewhat surprisingly the mean yield of A. flos-aquae when N was present (0.15 mm³ l^-1), was significantly less (P=5%) than in the absence of N (13.26 mm³l^-1).
• 3 The relative rates and duration of growth, the availability of P and N, and heterocyst frequency as an index of nitrogen fixation are considered in relation to the seasonal succession and dominance of these three principal cyanophyte members of the Lough Neagh phytoplankton.

     

Competition between the Cyanobacterium Limnothrix redekei and Some Spring Species of Diatoms under P-Limitation

The nutrient saturated growth and the competition between Limnothrix redekei and some spring diatoms for phosphate were studied in semi-continuous cultures at 10°C with a 6/18 h light/dark cycle and at 15 °C with a 12/12 light/dark cycle. Under these conditions in unialgal cultures, the maximum specific growth rate of Limnothrix redekei was two to three time lower than that of the diatoms. However, Limnothrix redekei was the superior competitor for phosphate under both conditions in mixed cultures. The diatoms in mixed cultures from each species with Limnothrix redekei were competitively displaced but at a different rate. This allowed the diatoms to be ranked in terms of their competitive ability for phosphate at 10°C as follows: Synedra acus > Asterionella formosa > Stephanodiscus minutulus > Nitzschia acicularis > Cyclotella meneghiniana > Stephanodiscus neoastrea > Stephanodiscus hantzschii. The competitive ability of Limnothrix redekei at 15 °C was increased and the rank order of diatoms slightly changed. The hypothesis that cyanobacteria are promoted by phosphate because they are inferior competitors for phosphate is incorrect for Limnothrix redekei.     

The influence of daylength,light intensity and temperature on the growth rates of planktonic blue-green algae

The in vitro growth rates under continuous light of the four dominant blue-green algae in Lough Neagh, Anabaena flos-aquae Bréb., Aphanizomenon flos-aquae Ralfs fa. gracile Lemm., Oscillatoria agardhii Gom. and Oscillatoria redekei van Goor were slower than in situ rates from Lough Neagh that had been corrected for hours of light received by the algae. However, by culturing on a 6: 18 light-dark cycle in vitro growth rates were obtained that were similar to the in situ rates. Under continuous light small species showed the fastest growth with Oscillatoria redekei the dominant species. However, this pattern was almost completely reversed under the light-dark cycle with Oscillatoria redekei only exhibiting the fastest growth rate under low light conditions. This observation showed agreement with Lough Neagh field data which showed that Oscillatoria redekei reached its maximum crop in April while the other three species were dominant during the summer months. Compared to the generally assumed high thermal tendency of blue-green algae the temperature maxima of the four species were low. No growth was observed at 35°C for any species while Anabaena flos-aquae was severely inhibited at 25°C.     

Density Gradient Centrifugation Compromises Bone Marrow Mononuclear Cell Yield

Bone marrow mononuclear cells (BMNCs) are widely used in regenerative medicine, but recent data suggests that the isolation of BMNCs by commonly used Ficoll-Paque density gradient centrifugation (DGC) causes significant cell loss and influences graft function. The objective of this study was to determine in an animal study whether and how Ficoll-Paque DGC affects the yield and composition of BMNCs compared to alternative isolation methods such as adjusted Percoll DGC or immunomagnetic separation of polymorphonuclear cells (PMNs). Each isolation procedure was confounded by a significant loss of BMNCs that was maximal after Ficoll-Paque DGC, moderate after adjusted Percoll DGC and least after immunomagnetic PMN depletion (25.6±5.8%, 51.5±2.3 and 72.3±6.7% recovery of total BMNCs in lysed bone marrow). Interestingly, proportions of BMNC subpopulations resembled those of lysed bone marrow indicating symmetric BMNC loss independent from the isolation protocol. Hematopoietic stem cell (HSC) content, determined by colony-forming units for granulocytes-macrophages (CFU-GM), was significantly reduced after Ficoll-Paque DGC compared to Percoll DGC and immunomagnetic PMN depletion. Finally, in a proof-of-concept study, we successfully applied the protocol for BMNC isolation by immunodepletion to fresh human bone marrow aspirates. Our findings indicate that the common method to isolate BMNCs in both preclinical and clinical research can be considerably improved by replacing Ficoll-Paque DGC with adapted Percoll DGC, or particularly by immunodepletion of PMNs.     

Population dynamics of Limnothrix redekei,Oscillatoria lanceaeformis,Planktothrix agardhii and Pseudanabaena limnetica (cyanobacteria) in a shallow hypertrophic lake (Spain)

Populations of Limnothrix redekei, Oscillatoria lanceaeformis, Planktothrix agardhii and Pseudanabaena limnetica were found in a hypertrophic, gravel-pit lake near Madrid (Spain), throughout a one year sampling at weekly intervals. Physico-chemical factors, phytoplankton biomass and net growth rates were measured. Oscillatoria lanceaeformis was only observed a few weeks, probably being related to phosphorus limitation. Planktothrix agardhii biomass was related to PhAR irradiances, light attenuation coefficient in the water, Brunt-Väisäla frequency and decrease of soluble reactive phosphorus. Limnothrix redekei and Pseudanabaena limnetica biomass values were related to a decrease of inorganic nitrogen and temperature. A different lag response of populations was observed in relation to the environmental features.     

The phycocyanin to chlorophyll {alpha} ratio and other cell components as indicators of nutrient limitation in two planktonic cyanobacteria subjected to low-light exposures

The cell composition of the planktonic cyanobacteria, Oscillatoriaagardhii (Gomont) and Oscillatona redekei (van Goor), was comparedfor cultures grown under nitrogen (N) and phosphorus (P) limitation,and light climates which were energy (E) limited (photoperiods3:21 and 6:18 light:dark (LD) and irradiances 12–153 µmolm^–2s^–1). Increases in carbohydrate/protein ratio(CHO/Prot) and declines in chlorophyll a (Cha) and phycocyanin(PC) resulted from N and P limitation. N-, P- and E-limitedcultures could be distinguished on the basis of P content andthe ratio of PC/Cha. The P content range of 0.1–0.55%of ash-free dry weight (AFDW) for P-limited cultures was lowerthan that for N- and E-limited cultures (0.56–2.2 %AFDW).Cultures limited by N were distinguishable from E-limited cellsby lower PC/Cha ratios, ranging from 0 to 4.08, compared to3.9–6.9 for E-limited cells. Under the 3:21 LD cycle,the minimum PC/Cha ratio of E-limited cells was 4.5. Increasesin the CHO/Prot ratios were proportional to the difference betweenthe nutrient-limited growth rate and the non-nutrient-limitedgrowth rate. A comparison of the composition of the two speciesshowed greater accumulation of carbohydrate by O.agardhii undernutrient-limiting conditions, but that O. redekei had higherlevels of protein, chlorophyll a and phycocyanin and, in theabsence of P limitation, higher P contents than O.agardhii.     

N2-fixing cyanobacteria: Why they do not become dominant in shallow hypertrophic lakes

Summary Phytoplankton species shifts and succession phenomenona in lakes of increasing trophic state were considered, using the basic information on the growth kinetics of the species involved. One of the most obvious signs of advanced eutrophication is the dominance of cyanobacteria (blue-green algae). Striking examples are the shallow, hypertrophic Dutch lakes The Veluwerandmeren (e.g., Wolderwijd and Veluwemeer), whereOscillatoria agardhii, a non-N₂-fixing cyanobacterium, has become dominant over the green algae, diatoms and N₂-fixing cyanobacteria (BERGER, 1975).We have studied the natural population ofO.agardhii during the growing season, by using physiological indicators, and could adduce that the natural population was successively growing under phosphorus, light, or nitrogen limitation (ZEVENBOOM and MUR, 1978a,b; ZEVENBOOMet al., 1982). One might expect that during the period of nitrogen limitation the N₂-fixing speciesAphanizomenon flos-aquae would be favoured and would be able to outgrow the nitrogen-limitedO.agardhii. However, in these lakes,A. flos-aquae was present only in few numbers and a succession fromO. agardhii toA. flos-aquae did not occur. Although field observations may give some indication, they cannot give decisive answers to the question which factor is triggering the observed species shifts and species dominance in natural waters. Such answers can only be obtained from growth kinetic and physiological data of the species involved. In our opinion, the most important factor to consider is the availability of light energy, which decreases with increasing eutrophication.The hypothesis was proposed by Mur and coworkers (MURet al., 1978) that in hypertrophic lakes the prevailing light conditions (low light irradiance) are more favourable forO.agardhii, since this species has a much lower requirement of light energy for growth than green algae as a consequence of its lower specific maintenance rate constant, _e (VAN LIERE, 1979; GONS, 1977). Competition experiments, performed withO. agardhii andScenedesmus protuberans under lightlimiting conditions, confirmed the hypothesis (MURet al., 1978), Continuous culture experiments withA. flos-aquae showed that also this species had a higher energy requirement thanO. agardhii (ZEVENBOOM, 1980). The differences were not found in the value of _e, but in the growth efficiency. The higher energy requirement ofA.flos-aquae was expected, since energy is needed for heterocyst production and N₂ fixation. Under light-limiting conditions and nutrient sufficiency (including nitrogen-nitrate) it can thus be expected that the N₂-fixer will be outcompeted by the non-N₂-fixing cyanobacterium. This was indeed observed (ZEVENBOOM et al., 1981).We further investigated the competitive interactions betweenA.flos-aquae, O. agardhii andS. protuberans under different sets of irradiance values and nitrate concentrations. We used the growth kinetic data of the species involved, which were obtained by means of continuous culture experiments (GONS, 1977; VAN LIERE. 1979; VAN LIERE and MUR, 1979; GONS and MUR, 1980; ZEVENBOOM and MUR, 1980; ZEVENBOOMet al., 1980; ZEVENBOOMet al., 1981). The competing species could be placed along the gradients of light irradiance values and nitrate concentrations, their positions being defined by their energy requirements and half-saturation constants for nitrate-limited growth, respectively. Distinct niches for the three species were found with respect to light and nitrate. Under conditions of low irradiance values and low (realistic) nitrate concentrations, nitrogen-limitedO.agardhii was able to outgrowA. flos-aquae andS. protuberans as a consequence of its low energy requirement and its high affinity for nitrate. The growth rates of the last two species were restricted by the limited availability of light. However, at high irradiance values,O.agardhii was inhibited in its growth rate and therefore failed to outgrow the other two species. The competition was then restricted to nitrogen-limitedS.protuberans and light-limitedA.flos-aquae; the latter could dominate at low nitrate concentrations. The results of competition experiments withO.agardhii andA.flos-aquae under different sets of irradiance values and nitrate concentrations agreed well with the niche-model described above (Zevenboom, unpubl. results).In conclusion, kinetic data of growth, obtained with continuous culture experiments, can provide basic information to explain species shifts and dominance in lakes with increasing eutrophication. Nitrogen-limiting conditions favour N₂-fixing cyanobacteria only when sufficient light is available for their growth (in less hypertrophic waters). The trophic state is thus of major importance and decisive with regard to which species will dominate.     

Growth, production and losses of phytoplankton in the lowland River Spree. I. Population dynamics

The seasonal and longitudinal dynamics of dominant phytoplanktonpopulations were observed in the lower Spree (Germany), a lowlandriver draining several lakes. Centric diatoms and cyanobacteriawere the chief components of the total phytoplankton biomassof 16 mg^–1 at the end of the river course (average March-October,1988–89). The cyanobacteria required a retention timeof several weeks in a drained lake to establish stable populations,whereas the other algal populations originated from upstreamimpoundments and slow-flowing river stretches. In spring, thedominant populations increased (the diatoms Nitzschia acicularis,Asterionella formosa, Stephanodiscus hantzschii. Cyclostephanosdubius, Cyclotella radiosa, C.meneghiniana) or kept their abundancesnearly constant (cyanobacterium Limnothrix redekei. diatom Synedraacus), but the biomass of the ‘summer species’ (diatomActinocyclus normanii, cyanobacteria Planktothrix agardhii andAphanizomenonsp.) was halved along the river course. These seasonal differencesin algal growth are probably caused by the changed mixing patternin the regions of origin and thus different pre-adaptation toturbulent mixing. Pronounced dear-water phases occurred regularlyat the end of May. They were intensified along the river coursedespite the lack of mesozooplankton. The dynamics of phytoplanktonwere reflected in the proportions of dissolved and particulatenutrients. The nutrient supply was always high enough to makelimitations of algal growth improbable.     

Polymixis of a shallow lake (Groβer Müggelsee,Berlin) and its influence on seasonal phytoplankton dynamics

The polymictic properties of Lake Müggelsee, a eutrophic shallow lake in Berlin, are described by the water column stability (N ²) and gradients in saturation of oxygen at the deepest site of the lake (7.5 m). Mixing and stratification changed irregularly up to 7 times during the vegetation season (April to September), as was indicated by all of the stratification parameters. Thermally stable conditions generally lasted 1–2 weeks. A maximum of 5 weeks stratification was observed in 1982.In order to investigate the response of algal development, the internal rates of change of the dominant algal species in the lake during the vegetation period were estimated from weekly measurements of phytoplankton biomass from 1980 to 1990. The necessity taking a mixed sample in a shallow lake is discussed. The polymictic properties favoured the development of specific blue-green algal species; there dominance was also favoured by the trophic conditions. Among the dominant blue-greens the growth of Limnothrix redekei was independent of polymixis whereas stratification supported the starting conditions for the summer blue-greens Aphanizomenon flos-aquae and Planktothrix agardhii. After these algae reached a distinct level of biomass, they grew under mixing as well as under stratified conditions.For the development of solitary centric diatoms during summer regulation by growth restriction through nutrient limitation, esp. dissolved silicon was more important. However, Melosira sp. developed well under stratified conditions but collapsed due to increased sinking losses when the water column became too stable.An attempt is made to apply Reynolds' possibility matrix of the most likely phytoplankton assemblages as a function of nutrients and mixing in the shallow Lake Müggelsee.     

Morphology and taxonomy of Oscillatoria redekei (Cyanophyta)

The polymorphous trichome apices of Oscillatoria redekei are described with respect to their shape and size. Breakage of trichomes may occur either trans- or intercellularly. The development and localization of intercellular trichome breakage were observed. Transmission electron microscopy shows that the fine structure of cell walls resembles that of Pseudanabaena galeata. Longitudinal walls consist of three or four layers and lack a sheath, while the cross walls are composed of three layers with a dominating L₂-layer. In contrast to P. galeata, the centroplasm and most of the thylakoidal region of adjacent cells of O. redekei are connected by the cross walls forming obtuse angles at the site of the longitudinal walls. The main characteristics of O. redekei and of Pseudanabaena species are discussed.     

Oscillatoria agardhii var. suspensa nov. var.

Zusammenfassung Oscillatoria agardhii var. suspensa n. var. ist ein Süßwasser-Planktont, der O. agardhii nahe steht, sich aber durch geringere Abmessungen und den Bau der Trichom-Enden unterscheidet. Sie wurde durch Waschen von anderen Organismen befreit, was bisher noch bei keiner Cyanophycee gelungen war.Die Art lebt im Wasser schwebend. Nur in alten Kulturen sammelt sie sich an der Oberfläche an. O. agardhii var. suspensa ist streng photo-autotroph und macht von organischen Nährstoffen keinen Gebrauch. Sie ist sogar sehr empfindlich gegen manche von ihnen, besonders gegen Glucose (aber nicht gegen andere Zucker), die schon in geringen Konzentrationen die Vermehrung unterdrückt.Die weißlich-grüne Farbe der Art wird durch die Pseudovacuolen bewirkt; der Chlorophyllgehalt ist aber nicht geringer als der von anderen Cyanophyceen, die tief blaugrün aussehen.

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Summary Oscillatoria agardhii var. suspensa n. var. is a freshwater planktont allied to O. agardhii but differing in smaller dimensions and the shape of the trichome-ends. It was freed from other organisms by the washing procedure, a technique that had not yet been successful in any member of the Cyanophyceae.The organism lives suspended in the water except in old cultures when it tends to accumulate near the surface. O. agardhii var. suspensa is strictly photo-autotrophic not utilizing any organic nutrients and even to some of them very sensitive, particularly to glucose (though not to other sugars) which prevents growth at minute concentrations.The whitish green hue of the species is due to pseudovacuoles while the content of chlorophyll is not lower than that of Cyanophycean species appearing deep blue-green to the eye.

     

Modification of littoral algal assemblages by gardening caddisfly larvae

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Chromatic Adaptation of the Planktonic Blue-green Alga Oscillatoria redekei VAN GOOR and its Ecological Significance

Natural populations and culture strains of Oscillatoria redekei isolated from the same lakes exhibit a great variety of phycoerythrine/phycocyanine ratios even under the same growth conditions. Cytophotometric and spectrophotometric determinations of the phycoerythrine and phycocyanine contents of different strains after exposure to blue-green and orange-red light, respectively, suggested that chromatic adaptation of O. redekei takes place both at cellular and population level. This wide intraspecific diversity is considered at least one reason for competitiveness in highly eutrophic lakes.     

The influence of flushing on nutrient dynamics,composition and densities of algae and transparency in Veluwemeer,The Netherlands

Total phosphorus and chlorophyll decreased significantly after reduction of the external phosphorus loading and the start of flushing Veluwemeer with polder water in 1979.Flushing of Veluwemeer has had a large impact on nutrient dynamics. Especially in the first winter, dilution was the main cause of changes in water quality. On a longer term the increase of the inactivation of phosphorus in sediments is important. Oscillatoria agardhii has been brought to the margins of its habitat. Three successive cold winters were an additional causal factor in the disappearance of Oscillatoria agardhii and the dominance of diatoms and green algae from 1985 onwards.Due to higher detritus and inorganic suspended matter concentrations transparency increased less than expected. Since 1985 chlorophyll only contributes for a small percentage to the transparency.In the present situation further improvement of the water quality of Veluwemeer is questionable, as the phosphorus concentration in the lake and the polder water is almost the same. Therefore it is recommanded to shift flushing operations, at least in the winter period, from Veluwemeer towards Wolderwijd.     

Membrane Populations of Bovine Choroid Plexus: Separation by Density Gradient Centrifugation in Modified Colloidal Silica

Abstract: The choroid plexus is intimately involved in the production and regulation of the cerebrospinal fluid. Populations of surface membranes from this epithelial tissue were separated by density gradient centrifugation by use of modified colloidal silica (Percoll). A fraction of heavy microsomes (P₃) containing plasma membranes was prepared by differential centrifugation. Membranes in fraction P₃ were mixed with a given concentration of Percoll and density gradients generated during centrifugation. When fraction P₃ was mixed with 20% (v/v) Percoll and centrifuged at 20,000 r.p.m. for 1 h in a 50.2 Ti fixed-angle rotor, membranes containing alkaline phosphatase (AP) were found at a density of 1.037 g/cm³ while those containing NaK ATPase were found at 1.047 g/cm³. With more shallow density gradients using 12% and 14% Percoll, a broad shoulder of AP activity became manifest at densities greater than 1.060 g/cm³ suggesting multiple populations of membranes containing AP. Membranes containing AP could also be separated from membranes containing γ-glutamyl transpeptidase (γ-GTP); this separation was most pronounced in 12% Percoll. The activity of γ-GTP could not be separated from activity of NaK ATPase. Total protein was distributed broadly throughout the gradients. Studies have been undertaken to compare the behavior of choroidal membranes in Percoll gradients with that of renal membranes because the biochemical anatomy of the kidney has been extensively studied. In contrast to choroidal membranes, renal membranes with NaK ATPase activity were found to have densities lower than those membranes with AP. Thus, the distribution of membrane-bound enzymes from kidney in a Percoll gradient was exactly the opposite of that observed for these same enzymes from choroid plexus. In addition, unlike the γ-GTP activity of choroid plexus, γ-GTP from kidney could be separated from the activities of both alkaline phosphatase and NaK ATPase. These marked differences in membrane populations between choroid plexus and kidney as defined by Percoll density gradient centrifugation analyses are presumably reflective of differences in the functions of the two epithelial tissues.     

Efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen

The objective was to compare four commercially available density gradient centrifugation (DGC) media (ISolate [Irvine Scientific; Santa Ana, CA, USA], Percoll [Pharmacia; Uppsala, Sweden], PureCeption [SAGE In-Vitro Fertilization, Inc.; Trumbull, CT, USA], PureSperm 100 [Nidacon International AB; Molndal, Sweden]) for their ability to separate viable, motile sperm from contaminant nonviable (immotile and/or dead) sperm and red blood cells (RBC). Pooled sperm-rich fractions from four healthy dogs were assessed using Spermvison SAR (Minitube of America). For this, 1 mL of the blood/sperm admixture was pipetted over 4 mL of DGC media: 50%/90% ISolate (Irvine Scientific), 45%/90% Percoll (Pharmacia), 40%/80% PureCeption (SAGE In-Vitro Fertilization, Inc.), and 40%/80% PureSperm 100 (Nidacon International AB). After centrifugation, five 1-mL fractions (A, B, C, D, and E) and the sperm pellet (bottom fraction F) were separated. Sperm morphology and red blood cell/sperm ratio (RBC/S) per fraction were determined on stained slides. All DGC media separated RBC from sperm; the highest red blood cell/sperm ratio was present in ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction A (29.4 ± 29.7 and 28.2 ± 20.8, respectively), and in fractions A and B of both PureCeption (SAGE In-Vitro Fertilization, Inc.) (37.0 ± 22.8 and 39.6 ± 24.3, respectively) and PureSperm 100 (Nidacon International AB) (25.2 ± 5.9 and 23.0 ± 3.9, respectively). The fractions with the highest total sperm recovery, motile sperm recovery, as well as overall motility were ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction D (33.9 ± 29.4%; 40.99 ± 27.9%; 71.2 ± 21.8% and 36.4 ± 14.5%; 39.3 ± 15.8%; 88.6 ± 2.3%, respectively), and for PureCeption (SAGE In-Vitro Fertilization, Inc.) and PureSperm 100 (Nidacon International AB), the sperm pellet, fraction F (78.8 ± 28.3%; 88.0 ± 17.4%; 70.2 ± 11.1% and 73.1 ± 21.0%; 75.4 ± 24.6%; 80.6 ± 17.1%, respectively). In the pellet for PureCeption (SAGE In-Vitro Fertilization, Inc.), more sperm and motile sperm were recovered than in ISolate (Irvine Scientific) and Percoll (Pharmacia) fractions D (P < 0.0163). Therefore, DGC media should be considered for canine semen purification when contaminated with blood or when separation of motile versus immotile sperm is needed.     

Use of Percoll density gradient centrifugation for preparing isolated rat hepatocytes having long-term viability

A method for preparing suspensions of hepatocytes with long-term viability is described. Its originality lies in the use, after conventional preparation, of a Percoll density gradient centrifugation which allows a complete and rapid separation of cell debris and released proteases from the intact hepatocytes. A series of functional reactions characterizing drug metabolism (P-450 level, ethoxycoumarin hydroxylation, methylumbelliferone conjugation) and membrane transport and integrity (α-aminoisobutyric acid transport and its stimulation by both insulin and glucagon) were conducted, in parallel to the trypan blue exclusion test, to evaluate the metabolic activity of both conventional and Percoll preparations. This investigation clearly demonstrates that the use of Percoll density gradient centrifugation significantly increases (from 5–6 to 30–35 h) the half-life of freshly isolated hepatocyte suspensions.     

Chemotaxonomy of planktonic cyanobacteria based on non-polar and 3-hydroxy fatty acid composition

Twenty-eight axenio planktonic cyanobacterial strains (10 Microcystis, three Oscillatoria, one Spirulina, one Aphanizomenon, 13 Anabaena) were investigated for their fatty acid composition by measurement of non-polar and hydroxy fatty acids. No 2-hydroxy fatty acids were detected in any strain, but 3-hydroxy fatty acids were detected in minor quantities in 24 strains. The highest portion of total fatty acids were non-polar fatty acids. Qualitative and quantitative analyses of 3-hydroxy fatty acids showed no taxonomic value in these strains, while the type of non-polar fatty acid composition was shown to be consistent within Microcystis and Anabaena strains, distinguishing them as type 4, characterized by the presence of 18:4, and type 2, characterized by 18:3 (α) of the Kenyon-Murata system. Two Oscillatoria agardhii Gomont strains were also included in the type 2 group due to the presence of 18: 3 (α), but the difference in characteristics of 16:2 and 16:3 between O. agardhii and Anabaena further divided type 2 into two subgroups: type 2A for Anabaena and type 2B for O. agardhii. A simplified unweighted pair group method with arithmetic averages (UPGMA) dendrogram demonstrated that the classification of 28 strains (Microcystis spp., Anabaena spp., Aphanizomenon flos-aquae (Lemmermann) Ralfs f. gracile (Lemmermann) Elenkin, O. agardhii and Spirullnasubsalsa Oersted ex Gomont based on numerical analysis of non-polar fatty acids corresponded to morphological species criteria, suggesting that non-polar fatty acid composition is a valuable chemical marker in the taxonomy of planktonic cyanobacteria. However, the fatty acid composition in Oscillatoria raciborskii is similar to that of Microcystis and very different from that of O. agardhii, suggesting its special position in Oscillatoria and the chemical diversity in the genus Oscillatoria.     

Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation

A procedure was developed to purify simultaneously peroxisomes and mitochondria from spinach (Spinacia oleracea L.) leaf under isoosmotic and low viscosity conditions. This method involved differential centrifugation and density gradient centrifugation on four layers of Percoll. Chlorophyll-free preparations of highly intact and active organelles were obtained and cross-contamination was negligible. Both organelles were stable for several hours, even if they remained in Percoll. Purified mitochondria were able to carry out the oxidation of different substrates with excellent respiratory control and ADP:O ratios. The method described in the present work was also suitable to purify mitochondria and peroxisomes from potato (Solanum tuberosum L.) tubers.