A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments

AIMS: The purpose of this study was to compare a recently described medium, thiosulphate-chloride-iodide (TCI), for the isolation of estuarine vibrios with thiosulphate-citrate-bile salts-sucrose (TCBS). METHODS: A total of 492 colonies which developed on these media from estuarine water samples taken monthly over a 10-month period were examined. RESULTS: A much larger number of colonies developed on TCBS than TCI, and minimal taxonomic criteria indicated that a higher percentage (61%) of TCBS colonies could be identified as Vibrio spp. when compared with TCI (46%). SIGNIFICANCE: This study suggests that TCBS is a superior medium when compared with TCI for the isolation of Vibrio spp. from estuarine waters. Because of the public health risk presented by V. vulnificus, V. parahaemolyticus, V. cholerae and other vibrios, the selection of the most appropriate medium for their isolation is extremely important.     

Method for Isolation and Purification of Cyanobacteria

A method employing nutrient saturated glass fiber filters allowed the isolation of the same numbers of cyanobacteria from freshwater as were obtained with medium solidified with agar, while providing a 2- to 15-fold reduction in the number of accompanying heterotrophic bacteria. Imipenem, a broad-spectrum β-lactam antibiotic which inhibits peptidoglycan biosynthesis, was superior to some other β-lactam antibiotics for reducing the numbers of heterotrophic bacterial contaminants associated with freshly isolated cyanobacteria to a level which facilitated the production of axenic cyanobacterial cultures.     

IN-SITU MORPHOLOGY AND OCCURRENCE OF EUCARYOTIC PHOTOTROPHS OF BACTERIAL SIZE IN THE PICOPLANKTON OF ESTUARINE AND OCEANIC WATERS1

Concentrates of the picoplankton (0.2–2.0 μm) sized fraction from the euphotic zone of estuarine and oceanic waters were examined by transmission electron microscopy. In addition to the numerous phototrophic procaryotes (chroococcoid cyanobacteria) previously reported, small phototrophic eucaryotes were observed in 20 of 25 samples examined. Micromonas pusilla (Butcher) Manton and Parks, a 1 × 1.5 μm flagellate, was abundant in estuarine samples in summer. Similar sized cells of non-flagellated chlorophytes, either Nannochloris Naumann or Chlorella Beijerinck, were observed sporadically in many samples. The most ubiquitous microalga was a scaled, non-flagellated prasinophyte that occurred at 9 of 15 different locations on 15 of 20 sampling dates in water samples from Iceland to the Caribbean Sea, This tiny alga (0.5 to 1.0 μm in diam.) is probably the smallest known photo-trophic eucaryote and has not heretofore been described. Enrichment cultures using conventional techniques on several cruises yielded only the Chlorella-type of green alga, as well as numerous isolates of unicellular chroococcoid cyanobacteria.     

RESPONSES OF THE PHYTOPLANKTON COMMUNITY GROWTH RATE TO NUTRIENT PULSES IN VARIABLE ESTUARINE ENVIRONMENTS

In estuaries, phytoplankton are exposed to rapidly changing conditions that may have profound effects on community structure and function. In these experiments, we evaluated the growth, productivity, and compositional responses of natural phytoplankton communities exposed to limiting nutrient additions and incubation conditions typical of estuarine habitats. Mesocosm bioassays were used to measure the short-term (2-day) growth rate, primary productivity, and group-specific biomass responses of the phytoplankton community in the Neuse River Estuary, North Carolina. A three-factor (mixing, sediment addition, and nutrient addition) experimental design was applied using 55-L mesocosm tanks. Growth rates were determined using the ¹⁴C photopigment radiolabeling method, and the abundance of algal groups was based on quantification of chemosystematic photopigments by HPLC. For Neuse River Estuary phytoplankton communities, stratified (nonmixed), turbid, and low-nitrate conditions favored increases in cryptomonad biomass. Mixed, turbid, high-nitrate conditions were favorable for increased primary productivity and chlorophytes, diatoms, and cyanobacteria. The highest community growth rates occurred under calm, high-nitrate conditions. This approach provided an assessment of the community-level phytoplankton responses and insights into the mechanisms driving blooms and bloom species in estuarine waters. The ability to rapidly alter growth rates to capitalize on conditions conducive for growth may play an important role in the timing, extent, and species involved with blooms in estuarine waters. Adaptive growth rate responses of individual species, as well as the community as a whole, further illustrate the sensitivity of estuarine ecosystems to excessive N inputs.     

An Improved Method for Marine Cyanobacterial DNA Isolation

Summary The method of Bolch, Blackburn, Jones, Orr & Grewe [Phycologia, 36, 6 11, 1997] developed for isolation of DNA from freshwater cyanobacteria was suitably modified to yield a simple, efficient and reproducible protocol for the isolation of DNA from different morphological types of marine cyanobacteria. This method resulted in a high yield of quality DNA suitable for polymerase chain reaction (PCR) amplifications.     

A selective and differential medium for Vibrio harveyi.

A new medium, termed Vibrio harveyi agar, has been developed for the isolation and enumeration of V. harveyi. It is possible to differentiate V. harveyi colonies from the colonies of strains representing 15 other Vibrio species with this medium. This medium has been shown to inhibit the growth of two strains of marine Pseudomonas spp. and two strains of marine Flavobacterium spp. but to allow the growth of Photobacterium strains. Colonies displaying typical V. harveyi morphology were isolated from the larval rearing water of a commercial prawn hatchery with V. harveyi agar as a primary isolation medium and were positively identified, by conventional tests, as V. harveyi. This agar displays great potential as a primary isolation medium and offers significant advantages over thiosulfate-citrate-bile salts-sucrose agar as a medium for differentiating V. harveyi from other marine and estuarine Vibrio species.     

Monitoring Heterocyst Isolation in Anabaena PCC 7119

The purification of isolated and intact heterocysts is an essential step in the study and characterisation of their specific proteins. Therefore, a method for very successful heterocyst isolation from filamentous cyanobacteria, as monitored by measuring the presence of ferredoxin-NADP⁺ reductase activity during the isolation procedure has been developed.This is an improvement over the current lysozyme method in which damage could be caused to the heterocysts septum releasing soluble proteins. Frozen filaments should not be used for heterocysts isolation.     

Fine-scale genetic structure, estuarine colonization and incipient speciation in the marine silverside fish Odontesthes argentinensis

The identification of incipient ecological species represents an opportunity to investigate current evolutionary process where adaptive divergence and reproductive isolation are associated. In this study we analysed the genetic structure of marine and estuarine populations of the silverside fish Odontesthes argentinensis using nine microsatellite loci and 396 bp of the mitochondrial DNA (mtDNA) control region. Our main objective was to investigate the relationship among estuarine colonization, divergent selection and speciation in silversides. Significant genetic structure was detected among all marine and estuarine populations. Despite the low phylogeographic structure in mtDNA haplotypes, there was clear signal of local radiations of haplotypes in more ancient populations. Divergence among marine populations was interpreted as a combined result of homing behaviour, isolation by distance and drift. On the other hand, ecological shifts due to the colonization of estuarine habitats seem to have promoted rapid adaptive divergence and reproductive isolation in estuarine populations, which were considered as incipient ecological species. This conclusion is supported by the existence of a set of environmental factors required for successful reproduction of estuarine ecotypes. The pattern of genetic structure indicates that phenotypic and reproductive divergence evolved in the face of potential gene flow between populations. We suggest that the 'divergence-with-gene-flow' model of speciation may account for the diversification of estuarine populations. The approach used can potentially identify 'incipient estuarine species', being relevant to the investigation of the evolutionary relationships of silversides in several coastal regions of the world.     

Pharmaceuticals from cultured algae

Summary An algae screening program, including cultured macroalgae, cultured cyanobacteria and cultured eukaryotic microalgae has been undertaken. Methods for the isolation, purification, preservation and cultivation of axenic cyanobacteria and eukaryotic cultures have been developed. Screening of these groups for biologically active components has lead to the isolation of pachydictyol and caulerpenyne from cultured macroalgae, while a series of hapalindoles and an antifungal depsipeptide have been isolated from cyanobacteria.     

A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture,mat and soil suitable for genomic fingerprinting and community analysis

This study presents a phenol and lysozyme free protocol for genomic DNA isolation of cyanobacteria from culture, mats and soil. For an efficient and pure DNA isolation from cyanobacteria having tough cell wall, extra steps of glass beading and Sepharose 4B purification were added. The modified method gave a higher yield of DNA than the phenol: chloroform extraction method. Four parameters selected for purity testing of the isolated DNA were: (i) restriction digestion with Hind III, (ii) randomly amplified polymorphic DNA-PCR of axenic culture of cyanobacteria to assess phylogenetic relatedness, (iii) denaturing gradient gel electrophoretic (DGGE) analysis of cyanobacterial mat and soil to ascertain the applicability of the isolated DNA for community analysis, and (iv) sequencing of partial 16S rDNA of Hapalosiphon intricatus BHULCR1, Anabaena doliolum LCR1, Anabaena oryzae LCR2, Aulosira fertilissima LCR4, and Tolypothrix tenuis LCR7 and BLAST analysis to confirm their cyanobacterial identity. Data generated from above analyses lead us to conclude that the modified method in question is rapid, cost effective, health and time conscious and promising for genetic fingerprinting and community analysis of cyanobacteria from diverse habitats.     

Evaluation of selective media for isolation and enumeration of vibrios from estuarine waters

We have compared the effectiveness of four media developed in the last years together with the medium GSTC(glucose-salt-tellurite-crystal violet), devised in our laboratory, for the recovery of vibrios from estuarine waters. In addition, a number of reference Vibrio and non-Vibrio strains have been tested for growth on the five media. TCBS and GSTC were the most selective media for reference strains of Vibrio spp. However, when the media were tested with samples of water from three different sites of an estuary, only TCBS was effective enough to recover vibrios inhibiting the growth of non-Vibrio populations. We also report here the usefulness of TCBS for isolation of the fish pathogen V. anguillarum, since a total of 81 strains isolated from diseased fish and water in various parts of the world grew on this medium. In conclusion, we consider the TCBS as the best medium to isolate Vibrio species pathogenic for humans and fish, and for recovery of vibrios from estuarine waters.     

Modern methods for isolation,purification, and cultivation of soil cyanobacteria

Up-to-date methods for isolation of cyanobacteria from soil samples, removal of accompanying microflora, obtaining axenic strains, and conditions and media for subsequent cultivation are reviewed. Characterization of soil as a specific habitat for cyanobacteria is provided. Comparative analysis of pH and elemental composition of the liquid phase of most soil types with the media for cultivating cyanobacteria is carried out. The functional role of the major components required for the cultivation of cyanobacteria is described. The problems associated with isolation, purification, and cultivation of soil cyanobacteria, as well as the relevant solutions, are discussed.     

An effective method based on wet-heat treatment for the selective isolation of Micromonospora from estuarine sediments

Several methods for the isolation of Micromonospora from soil samples have been developed; however, it is unclear whether these methods are optimal for estuarine samples. In this study, we optimized the conditions of a wet-heat method for the selective isolation of Micromonospora from estuarine sediments. Sediments were collected from the Arakawa River (estuarine sediments) and Tokyo Bay (marine sediments). Sediment samples were wet-heated at 45, 55, or 65 °C for 30 min and then incubated at 27 °C for 3 weeks. After incubation, most of the actinomycete colonies were macroscopically determined to be of the genus Micromonospora or Streptomyces. In contrast to the treatment at 55 °C, treatment at 65 °C drastically reduced the number of Streptomyces colonies but increased the number of Micromonospora colonies from the estuarine sediments. This procedure allowed us to grow cultures that were composed of more than 90 % Micromonospora. In addition, treatment at 65 °C did not affect the diversity of Micromonospora species compared with treatment at 55 °C. These results indicate that the wet-heat method, which involves pre-treating the sediment at 65 °C for 30 min, is a very simple and effective method for the selective enrichment of a large number of diverse Micromonospora from estuarine sediments. Our results may lead to the isolation of new Micromonospora species, which produce novel bioactive compounds, from different estuarine sediments.     

A review of microcystin detections in Estuarine and Marine waters: Environmental implications and human health risk

Toxin production by harmful cyanobacteria blooms (CyanoHABs) constitutes a major, worldwide environmental threat to freshwater aquatic resources that is expected to expand in scale and intensity with global climate change. Extensive literature exists on the most frequently encountered cyanotoxin, microcystin, in freshwater environments. Yet, the expansion of microcystin producing CyanoHABs and the transport of contaminated inland waters to estuarine and coastal marine waters has only recently received attention. This paper synthesizes information on the salinity tolerance of microcystin producing cyanobacteria and summarizes available case reports on microcystin presence in estuarine and coastal waters. We highlight a potential food-borne exposure route to humans by reviewing the growing body of evidence that shows microcystins can accumulate in coastal seafood. These cases reinforce the importance of freshwater nutrient reduction and the need for freshwater management efforts to look beyond lacustrine and riverine systems. Events reviewed here likely only represent a small proportion of cases where microcystins affect estuarine and coastal waters. We strongly suggest increased monitoring and research efforts to understand, react to, and prevent ecological and health problems associated with the growing problem of toxic CyanoHABs in coastal environments.     

Optimal enrichment time for isolation of Vibrio parahaemolyticus from seafood.

The growth of Vibrio parahaemolyticus in a liquid medium was compared with that of human fecal flora and estuarine flora. No marked differences were noted between growth at 25 and 37 degrees C for V. parahaemolyticus. However, the marine organisms were strongly inhibited when incubated at 37 degrees C. Incubation for 8 h in an enrichment broth yielded V. parahaemolyticus growth, even with a small inoculum, whereas the marine and fecal floras were inhibited. Therefore, enrichment for 8 h at 37 degrees C appears to be optimal for isolation of V. parahaemolyticus, permitting more rapid results in seafood analysis.     

A cyanobacterial recombination study, involving an efficient N2-fixing non-heterocystous partner

Many filamentous cyanobacteria fix atmospheric nitrogen under natural conditions in specialized anaerobic compartments, heterocysts, interspersed between vegetative cells, which provide protection to the O2-sensitive nitrogenase. A few unicellular cyanobacterial strains are also known to fix nitrogen aerobically at a slower rate. Filamentous cyanobacteria lacking heterocysts are not known so far to fix nitrogen. We describe the isolation and purification of a non-heterocystous filamentous cyanobacterium from the fronds of the water-fern Azolla, fixing nitrogen at 18.7+/-0.2 n moles ethylene microg Chl. a(-1) h(-1) when grown in nitrogen-free medium at a low level of oxygen between two layers of agar. This strain of Anabaena azollae has been designated as het- nif+ (non-heterocystous and nitrogen-fixing), and is found to be easily and effectively preserved in nitrogen-free medium in standard synthetic cyanobacterial nutrient medium (pH 8.5) at a continuous light intensity of 2800 lx at 25+/-1 degrees C. This het- nif+ strain is an effective donor of the nif+ marker to a het+ nif- strain of another cyanobacterium, Nostoc muscorum, when both are grown together in a recombination study.     

Toxic effects of microcystins in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda,Grapsidae)

Microcystins are toxins produced by cyanobacteria, being toxic to aquatic fauna. It was evaluated alternative mechanisms of microcystins toxicity, including oxidative stress and histopathology in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Grapsidae). Microcystins was administered to crabs (MIC group) over 1 week, whereas the control (CTR group) received the saline from cyanobacteria culture medium. At day 7, catalase activity was higher in the MIC than in the CTR group, although a decrease of activity was verified in both groups with respect to time 0. Glutathione-S-transferase activity augmented in MIC with respect to CTR, suggesting a higher conjugation rate of the toxins with glutathione. No differences were detected in the superoxide dismutase activity. Lipid peroxidation remained stable in both groups. Histopathological analyses showed that the number of B cells decreased significantly in the CTR as a possible effect of starvation, while no significant change was observed in the MIC group. The hepatopancreas from the MIC group exhibited some necrotic tubules and melanin-like deposits. Overall, results showed that some enzymes of the antioxidant defense system were activated after microcystins exposure, this response being able to maintain lipid peroxidation levels, but insufficient to completely prevent histological damage.     

Quantification of microcystin-producing cyanobacteria and E. coli in water by 5'-nuclease PCR

AIMS: 5'-Nuclease (real-time, quantitative) PCR methodologies were developed and applied as diagnostic tools for the detection of microcystin-producing cyanobacteria and Escherichia coli in water. METHODS AND RESULTS: PCR was used to detect regions of the lacZ gene in E. coli, and the microcystin synthetase gene in microcystin-producing cyanobacteria. In environmental water samples, natural inhibitors to PCR were effectively removed with a prefiltration step and an EDTA wash. A lower detection limit of 10 cells ml(-1) was obtained with endpoint PCR detection. 5'-Nuclease PCR was used for microbial quantification of 1 ml inoculated water samples. We were able to detect down to three copies of our target genes per sample within about 2 h (post-DNA isolation) for both E. coli and microcystin-producing cyanobacteria. CONCLUSIONS: 5'-Nuclease PCR offers a rapid and sensitive method of bacterial quantification in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: 5'-Nuclease PCR can be adopted as an effective diagnostic tool for monitoring microbiological water quality, through coliform quantification, and detection of other waterborne microbial pathogens.     

INDUCTION OF VACUOLATED SPHEROPLASTS AND ISOLATION OF VACUOLES IN CYANOBACTERIA1

There is still a great deal of debate about whether cyanobacteria contain vacuoles. This might in part reflect our limited ability to isolate vacuoles. We found and isolated vacuoles from different cyanobacteria during spheroplast preparation. Lysozyme treatment induced two kinds of spheroplasts: vacuolated spheroplasts and nonvacuolated spheroplasts. Upon breakage in distilled water, vacuolated spheroplasts released transparent, spherical, and colorless vacuoles with diameters ranging from 2.3 to 16 μm. Large vacuoles could be generated by fusion of two or three small vacuoles. Additionally, large vacuoles also could engulf small ones or other cellular bodies. The isolated vacuoles could tolerate hypotonic condition, and some could be drawn into a thread. Nonvacuolated spheroplasts released few vacuoles after breaking apart. This successful confirmation and isolation of vacuoles will allow studies of the origin and function of cyanobacterial vacuoles.     

紫色非硫细菌质粒的制备与性质

介绍一种适于紫色非硫细菌质粒的制备方法,该法简单,易操作,质粒ＤＮＡ纯度好,收率高,通过在加富培养基上连续传代数次,质粒ＤＮＡ可自行消除。