Cells In Laboratory Cultures Research Articles

Characterizing cell surface of blooming Microcystis in Lake Taihu, China.

Microcystis occurs as colonies in the natural environment but disaggregates into single cells in laboratory cultures. In order to explore the mechanism of how Microcystis forms colonies, the zeta potentials of Microcystis cells from the laboratory and the field were studied, and the hydrophobicity of Microcystis colonies in different sizes was investigated in Lake Taihu. The incubation experiment indicated that the zeta potentials of Microcystis cells were affected by growth phase and species. The absolute values in exponential phase were lower than those in stationary phase, suggesting that the cells with rapid growth easily formed colonies due to more instability on the cell surface. The values of Microcystis aeruginosa were higher than those of Microcystis flos-aquae, which confirmed that M. aeruginosa prevailed in waters for a longer time and at a larger size compared with M. flos-aquae. In another aspect, the absolute zeta potentials of Microcystis spp. at pH 7.0 decreased from spring to autumn in the field; the values in spring were higher than those in summer, suggesting that a large-sized Microcystis colony would more easily form in summer. Additionally, differences in hydrophobicity exist among Microcystis colonies of various sizes. The surface hydrophobicity of colonies in the <20 μm size class was higher than that of larger colonies. This characteristic allowed small colonies to easily form large colonies to survive better. These results would be helpful to understand the mechanism of the bloom formation, especially the colony formation, in Microcystis.

Experimental evidence for the role of heterotrophic bacteria in the formation of Microcystis colonies

Microcystis, a genus of cyanobacteria that is dominant in eutrophic lakes, occurs mainly as colonial morphs under natural conditions but as single cells in laboratory cultures. Recent studies have suggested that Microcystis–bacteria interactions significantly influence Microcystis morphology, but the underlying mechanism remains unclear. In this study, a total of 48 strains of heterotrophic bacteria were purified from Microcystis mucilage. Five bacteria, Aeromonas veronii, Enterobacter aerogenes, Exiguobacterium acetylicum, Bacillus cereus and Shewanella putrefaciens, can induce unicellular Microcystis to form colonies. Heterotrophic bacteria stimulated Microcystis growth and induced the production of extracellular polymeric substances in coculture treatments. Extracellular polymeric substances, such as extracellular polysaccharides (EPS), were responsible for the mucilage formation in colonial Microcystis. We analysed extracellular metabolic compounds produced by Microcystis aeruginosa and Microcystis wesenbergii using gas chromatography mass spectrometry. Filtrate extracts from coculture treatments indicated that some compounds, such as 2-dodecen-1-yl(-) succinic anhydride and benzoic acid, 2,3-bis[(trimethylsilyl)oxy]-, trimethylsilyl ester, might play a significant role in colonial M. aeruginosa or M. wesenbergii formation. Our data suggested that the interaction of Microcystis and heterotrophic bacteria was crucial for the formation of Microcystis colony and outbreak of Microcystis blooms.

Effect of fluid motion on colony formation in Microcystis aeruginosa

Microcystis aeruginosa, generally occurring in large colonies under natural conditions, mainly exists as single cells in laboratory cultures. The mechanisms involved in colony formation in Microcystis aeruginosa and their roles in algal blooms remain unknown. In this study, based on previous research findings that fluid motion may stimulate the colony formation in green algae, culture experiments were conducted under axenic conditions in a circular water chamber where the flow rate, temperature, light, and nutrients were controlled. The number of cells of Microcystis aeruginosa, the number of cells per colony, and the colonial characteristics in various growth phases were observed and measured. The results indicated that the colony formation in Microcystis aeruginosa, which was not observed under stagnant conditions, was evident when there was fluid motion, with the number of cells per largest colony reaching 120 and the proportion of the number of cells in colonial form to the total number of cells and the mean number of cells per colony reaching their peak values at a flow rate of 35 cm/s. Based on the analysis of colony formation process, fluid motion stimulates the colony formation in Microcystis aeruginosa in the lag growth phase, while flushes and disaggregates the colonies in the exponential growth phase. The stimulation effect in the lag growth phase may be attributable to the involvement of fluid motion in a series of physiological processes, including the uptake of trace elements and the synthesis and secretion of polysaccharides. In addition, the experimental groups exhibiting typical colonial characteristics in the lag growth phase were found to have higher cell biomass in the later phase.

Biochemical, morphological, and genetic variations in Microcystis aeruginosa due to colony disaggregation

Microcystis aeruginosa commonly occurs as large colonial morph under natural conditions, but disaggregates and exists as single cells in laboratory cultures. To demonstrate the adaptive changes, differentiation of carbohydrates, pigments, and protein between colonial and disaggregated M. aeruginosa were examined. Their morphological and ultrastructural characteristics were subsequently observed by scanning electron microscopy and transmission electron microscopy. Results showed that chlorophyll a and phycocyanin in cells, soluble carbohydrate produced in the culture medium, and total carbohydrate in cells and sheath of colonial M. aeruginosa are significantly higher (p 0.05) between them. Their morphological and ultrastructural characteristics were evidently different, and by morphological criteria they could be separated into two morphotypes. In addition, the genetic diversity of 16S–23S internal transcribed spacer of them were examined and compared with reference strains of M. aeruginosa. The alignment of two sequences revealed that genetic identity level was extremely high (96.94%) and no significant difference was found in the nucleotide diversity (0.014 ± 0.008). This suggested that similar genotypes could present distinct morphotypes in M. aeruginosa. The tree topologies and analysis of molecular variance of the two sequences and reference sequences from GenBank database indicated that the genotypes of M. aeruginosa strains were not always related to their localities and exhibit heterogeneity within a species.

Observation on colony formation of Microcystis aeruginosa induced by filtered lake water under laboratory conditions

Microcystis aeruginosa mainly occurs as single cells in laboratory cultures, but in the colonial morph under natural conditions. This phenomenon suggests that some factors may be responsible for the typical occurrence of colonies in lakes. To demonstrate this possible effect, lake water with abundant or few zooplankton was filtered through a 0.15 µm membrane filter, and used as test water. Microcystis aeruginosa was cultured in BG-11 medium, with or without test water. The results showed that filtered lake water with abundant zooplankton could induce colony formation in M. aeruginosa, whereas M. aeruginosa populations in the control and the treatment of filtered lake water with few zooplankton were still strongly dominated by unicells and paired cells, and no colony was formed. In the treatment of filtered lake water with abundant zooplankton, some colonies of several, dozens, and sometimes even hundreds of M. aeruginosa cells were formed. The unicells reduced to 53.4±6.5% and the proportion of cells in colonies increased from 0 to 24.5±4.6% of the populations. The mean number of cells per unit of M. aeruginosa in the treatment of filtered lake water with abundant zooplankton was promoted significantly compared with those of the control and the treatment of filtrated lake water with few zooplankton. In addition, no significant difference in growth rate was detected between the control and the treatments, regardless to the filtered lake water (i.e. with abundant zooplankton or with few zooplankton). Thus, we concluded that colony formation in our experiment was probably not associated with the nutrition difference but with some dissolved substances probably released from abundant zooplankton in the natural lake water, which may be one of the environmental factors responsible for the colonial form in M. aeruginosa.

Estimating abundance and single-cell characteristics of respiring bacteria via the redox dye CTC

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is used in aquatic sciences as a vital stain for enumeration of respiring bacteria in situ. Questions concerning its efficacy have been raised. We propose that the abundance of CTC-positive (CTC+) bacteria is a useful parameter in microbial ecology based on the following information: (1) Taxonomically diverse strains of aerobic, heterotrophic marine bacteria reduce CTC to its fluorescent product. (2) The proportion of CTC+ cells in laboratory cultures and in bacterioplankton assemblages varies in meaningful ways: the proportion of CTC+ cells is greatest for bacteria in log-phase growth, and lowest for bacteria in late stationary phase; particle-associated bacteria in various marine environments exhibit a higher percentage of CTC+ ceUs compared to bacteria freely suspended in the water column; the proportion of CTC+ cells in a bacterioplankton assemblage can be increased by an order of magnitude or more by addition of substrate, in the absence of net change in bacterial numbers. (3) Flow cytometric analysis shows a strong relationship between characteristics of CTC+ cells (abundance, size, red fluorescence) and rates of leucine incorporation by bacterial assemblages. We suggest that CTC+ cells represent those bacteria characterized by a high level of metabolic activity, and that cells which show no apparent reduction of CTC have either low or no metabolic activity. Some portion of CTC-negative (CTC-) cells may have sufficient RNA content, andlor ability to assimilate labile substrates at dilute concentrations, to be identifiable as 'active' via indices of cell-specific rRNA content or of rnicroautoradiography. Quantitative differences in metabolism between 'highly active' CTC+ cells, and 'less active' CTCcells have yet to be determined.

Phaeocystis globosa (Prymnesiophyceae) colonies: hollow structures built with small amounts of polysaccharides

In both field samples and cultures the total amount of sugar and of carbon in colonies of Phaeocystis globosa was correlated with colony surface area, suggesting a hollow structure. A conceptual model based on biochemical data and on the assumption that the mucus occurs as a layer of a fixed thickness, irrespective of colony size, predicts that the thickness of the layer is 7 μm. A confocal laser scanning microscope image of fluorescently labelled mucus confirmed this view of the colony structure. The measured contents of carbon and sugar per cell (including mucus) were constant for all colony sizes. Cells in laboratory cultures contained 122 pg C, which is twice the value for cells obtained at a field station in the North Sea (57 pg). In contrast, sugar per cell was higher in the field than in exponentially growing cultures. Therefore the percentage of sugar carbon relative to total carbon of colonies in the field was higher (19–35%) than that in cultures (10%). Highest values were found at low ambient nutrient concentrations, probably due to the presence of storage glucans in the cells that are produced under nutrient limitation. The functional role of Phaeocystis colonies in the pelagic system has to be re-evaluated because (i) biomass estimates have previously often been based on the assumption of a colony filled with mucus while in reality it is a hollow structure with an aqueous lumen, and (ii) in contrast to earlier reports it is shown that only a small amount of carbohydrate is needed to build the colony structure.

Phaeocystis globosa (Prymnesiophyceae) colonies : hollow structures built with small amounts of polysaccharides

In both field samples and cultures the total amount of sugar and of carbon in colonies of Phaeocystis globosa was correlated with colony surface area, suggesting a hollow structure. A conceptual model based on biochemical data and on the assumption that the mucus occurs as a layer of a fixed thickness, irrespective of colony size, predicts that the thickness of the layer is 7 μm. A confocal laser scanning microscope image of fluorescently labelled mucus confirmed this view of the colony structure. The measured contents of carbon and sugar per cell (including mucus) were constant for all colony sizes. Cells in laboratory cultures contained 122 pg C, which is twice the value for cells obtained at a field station in the North Sea (57 pg). In contrast, sugar per cell was higher in the field than in exponentially growing cultures. Therefore the percentage of sugar carbon relative to total carbon of colonies in the field was higher (19–35%) than that in cultures (10%). Highest values were found at low ambient nutrient concentrations, probably due to the presence of storage glucans in the cells that are produced under nutrient limitation. The functional role of Phaeocystis colonies in the pelagic system has to be re-evaluated because (i) biomass estimates have previously often been based on the assumption of a colony filled with mucus while in reality it is a hollow structure with an aqueous lumen, and (ii) in contrast to earlier reports it is shown that only a small amount of carbohydrate is needed to build the colony structure.

Human Papillomavirus and Human Disease

Human papillomaviruses (HPVs) are associated with a spectrum of different diseases in humans, including common warts and genital warts. Of more serious concern is the connection between certain HPV types and some malignancies, particularly cervical and anal cancer. DNA from HPV-16 and HPV-18, two types frequently found in cervical cancer tissue, can immortalize cells in laboratory cultures, unlike DNA from HPV types associated with benign genital lesions. Although it is unclear how high-risk HPV types cause cancer, studies indicate that malignant transformation involves the viral E6 and E7 gene products, which may exert their effect by interfering with the cellular proteins that regulate cell growth. The vast majority of those infected do not develop malignancies, indicating that HPV infection alone is not enough to cause cancer. Cofactors such as cigarette smoking, may be required before neoplasia can occur. The potential seriousness of HPV infections is suggested by the observations that the number of genital HPV infections diagnosed is increasing and that cervical cancer is the second leading cause of cancer deaths in women throughout the world.

THE ULTRASTRUCTURE AND CYTOCHEMISTRY OF RESTING CELL FORMATION INAMPHORA COFFAEFORMIS(BACILLARIOPHYCEAE)1

SUMMARYThe ultrastructure of logarithmic‐growing cells and of resting cells in laboratory cultures ofAmphora coffaeformis(Ag.) Kütz. isolated from deep ocean water was examined using electron and light microscopy. The acid Phosphatase activity, chlorophyll a and lipid content were assessed at weekly intervals of resting cell formation during cold‐dark treatment, simulating deep ocean water. Approximately 4 wk are required to complete resting cell formation. During the first week, the cytoplasm undergoes extensive transformation and lysosomal activity is observed. Large vacules decrease in size and many small ones develop, the mitochondria become fewer and one or more massive mitochondria appear possibly by fusion of smaller ones; the cytoplasm becomes densely granular. During the second and third week, the cytoplasm continues to contract, lipid bodies begin to develop and the plastid becomes densely stained. At the fourth week, the mature resting cell is formed containing one or more massive mitochondria, a well‐formed plastid, and granular cytoplasm containing occasional lipid droplets. There is no change in frustule morphology and the cytoplasm does not produce a protective layer. The variation in chemical constituents correlates with microscopic structure of the cells. The fine structure of cells during growth resumption when exposed to light at 25 c is presented. Previous reports of viable, chlorophyll‐containing cells at great depths in the ocean may be explained by the results reported in this paper.