Life cycle of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides in Korean coastal waters

Since 1995, blooms of the harmful dinoflagellate, Cochlodinium polykrikoides, have caused considerable mortality of aquatic organisms and economic loss in Korea. However, little is known about the life cycle of the species, except for the planktonic vegetative stage; therefore, the aim of this paper was to elucidate the life cycle of C. polykrikoides. Its life cycle has two morphologically different stages: an armored and an unarmored vegetative stage. Armored vegetative cells were found in seawater samples collected in late-November and developed into four-cell chained, unarmored vegetative cells under laboratory culture. In samples collected in late-May, both the armored and unarmored types (vegetative swimming stage) occurred; the former easily developed into an unarmored vegetative cell type, suggesting that the armoured–unarmored transition occurs as early as May. A presumptive resting cyst, round but folded at one side, was produced from armored type cells in laboratory conditions. It was also collected from natural bottom sediments, which suggests it is the dormant resting cyst of C. polykrikoides.     

Efficacy of three commercially available ballast water biocides against vegetative microalgae, dinoflagellate cysts and bacteria

One proposed solution to the problem of ballast-mediated aquatic invasions involves chemically treating ballast water to kill key target organisms. Here, we examine the efficacy of three commercially available ballast water biocides using vegetative microalgae, dinoflagellate resting cysts and bacteria as test organisms. Chemicals tested were the ballast water biocides SeaKleen^® and Peraclean^® Ocean, and the chlorine dioxide biocide Vibrex^®. Results demonstrate that the applicability of each of the three chemical biocides as a routine ballast water treatment is limited by factors such as cost, biological effectiveness and possible residual toxicity of the discharged ballast water (assessed on the basis of impact on motility of vegetative marine microalgae). Of the three biocides tested, Peraclean^® Ocean appears to hold the most potential; however its effectiveness in shipboard trials is yet to be proven. Peraclean^® Ocean was biodegradable within 2–6 weeks (initial concentration of 200 ppm), could effectively inactivate resting cysts of the marine dinoflagellates Gymnodinium catenatum, Alexandrium catenella and Protoceratium reticulatum at 400 ppm, could control bacterial growth of Escherichia coli, Staphylococcus aureus, Listeria innocua and Vibrio alginolyticus at 125–250 ppm, and could eliminate vegetative dinoflagellate cells at a concentration of 100 ppm. SeaKleen^® eliminated vegetative microalgae at 2 ppm and could control resting cysts of the dinoflagellates G. catenatum and P. reticulatum at a concentration of 6 and 10 ppm, respectively, when exposed for a period of 2 weeks. SeaKleen^® did not inactivate resting cysts of A. catenella at a concentration of 10 ppm and was found to degrade at a rate that could result in the discharge of residual toxic water into the marine environment. Together with the poor bactericidal properties of SeaKleen^® (100–200 ppm required), this may limit the use of this biocide as a routine treatment option. Vibrex^® is not a suitable ballast water treatment option due to the need for hydrochloric acid as an activator, however it was found to be the most effective against bacteria (complete inhibition at 15 ppm) indicating that onboard chlorine dioxide generators may provide an effective bacterial treatment option. The performance of these biocides was adversely influenced by a variety of factors including low water temperatures (6 °C compared to 17 °C), light versus dark conditions, and the presence of humus-rich seawater and ballast water sediments.     

Survival of coliforms and bacterial pathogens within protozoa during chlorination.

The susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria. Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were greater than 50-fold more resistant to free chlorine when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for survival of fastidious bacteria in dilute and inhospitable aquatic environments.     

Inactivation of Listeria monocytogenes/Flavobacterium spp. biofilms using chlorine: impact of substrate,pH, time and concentration

AIMS: To determine the effect of chlorine on mixed bacterial biofilms on stainless steel (SS) and conveyor belt surfaces. METHODS AND RESULTS: Biofilms were exposed to pH-adjusted (6.5) and non-pH-adjusted solutions of chlorine (200, 400 and 600 ppm) for either 2, 10 or 20 min and survivors enumerated. There were significant differences in cell death relating to chlorine concentration and exposure time for the cells attached to the SS, with solutions adjusted to pH 6.5 being more effective at reducing numbers. In contrast, on conveyor belt surfaces cell numbers decreased by less than two logs after 20 min regardless of treatment. CONCLUSIONS: Chlorine effectiveness is dependent on its concentration, solution pH, exposure time, the nature of the surface and the microbial species present. SIGNIFICANCE AND IMPACT OF THE STUDY the interests of food safety it is important that sanitizer users are aware of the conditions that effect their performance.     

Factors influencing attachment of thermophilic bacilli to stainless steel

AIMS: This project aimed to investigate the mechanism of attachment of the vegetative cells and spores of thermophilic bacilli to stainless steel with a view to devising strategies to limit biofilm development and survival. METHODS AND RESULTS: Spores and vegetative cells of bacterial isolates were exposed to protein denaturing agents (sodium dodecyl sulphate (SDS) and trypsin) and polysaccharide removing agents (sodium metaperiodate, trichloroacetic acid (TCA) and lysozyme). Treatment with sodium metaperiodate, TCA and lysozyme increased the number of vegetative cells attaching in many of the strains studied, while SDS and trypsin decreased attachment. Spores attached to stainless steel in greater numbers than vegetative cells, and the various treatments had less effect on this attachment than for vegetative cells. Viability of the cells or spores was not an important factor in attachment, as cells and spores rendered non-viable also attached to stainless steel in similar numbers. Coating the stainless steel with skim milk proteins decreased the attachment of both vegetative cells and spores. There was no correlation between the degree of attachment and the amount of extracellular polysaccharide (EPS) produced by each strain, surface hydrophobicity or zeta potential of vegetative cells or spores, though spores were found to be more hydrophobic than vegetative cells. CONCLUSIONS: The results suggest that biofilm formation by these thermophilic bacilli is probably a multifactorial process, and that cell-surface proteins play a very important role in the initial process of attachment during the formation of biofilms by these bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This information will provide direction for developing improved cleaning systems to control biofilms of thermophilic bacilli in dairy manufacturing plants.     

ULTRASTRUCTURE OF GLOEODINIUM MONTANUM (DINOPHYCEAE)1

The freshwater dinoflagellate Gloeodinium montanum Klebs (1912) was examined with transmission and scanning electron microscopy. Micrographs of ultrathin sections revealed a series of membrane layers rather than the usual dinoflagellate theca in vegetative cysts and in legates. Swarmers had distinct pellicles but appeared to be devoid of thecal plates and vesicles. The organization of cysts and swarmers appeared remarkably similar. All cell types had typical dinoflagellate nuclei with condensed chromosomes. Chloraplasts had girdle lamellae. One pyrenoid per cell was also present in chloroplasts of vegetative cysts. Starch grains and oil globules were distributed throughout the cytoplasm. Large accumulation bodies and polyvesicular vacuoles were found in aging cysts. Trichocysts and flagellar hairs were absent. Two types of intra-cellular prokaryotic organisms were discovered.     

A single-cell view of ammonium assimilation in coral–dinoflagellate symbiosis

Assimilation of inorganic nitrogen from nutrient-poor tropical seas is an essential challenge for the endosymbiosis between reef-building corals and dinoflagellates. Despite the clear evidence that reef-building corals can use ammonium as inorganic nitrogen source, the dynamics and precise roles of host and symbionts in this fundamental process remain unclear. Here, we combine high spatial resolution ion microprobe imaging (NanoSIMS) and pulse-chase isotopic labeling in order to track the dynamics of ammonium incorporation within the intact symbiosis between the reef-building coral Acropora aspera and its dinoflagellate symbionts. We demonstrate that both dinoflagellate and animal cells have the capacity to rapidly fix nitrogen from seawater enriched in ammonium (in less than one hour). Further, by establishing the relative strengths of the capability to assimilate nitrogen for each cell compartment, we infer that dinoflagellate symbionts can fix 14 to 23 times more nitrogen than their coral host cells in response to a sudden pulse of ammonium-enriched seawater. Given the importance of nitrogen in cell maintenance, growth and functioning, the capability to fix ammonium from seawater into the symbiotic system may be a key component of coral nutrition. Interestingly, this metabolic response appears to be triggered rapidly by episodic nitrogen availability. The methods and results presented in this study open up for the exploration of dynamics and spatial patterns associated with metabolic activities and nutritional interactions in a multitude of organisms that live in symbiotic relationships.     

Inactivation of indicator micro-organisms from various sources of faecal contamination in seawater and freshwater

AIM: The survival of indicator micro-organisms in aquatic systems is affected by both biotic and abiotic factors. Much of the past research on this topic has been conducted using laboratory-generated cultures of indicator bacteria. For this study, we used natural sources of faecal contamination as inoculants into environmental water samples, thereby representing the wide diversity of organisms likely to be found in faecal contamination. METHODS AND RESULTS: Rates of inactivation of water quality indicators, total coliforms (TC), Escherichia coli, enterococci (EC) and F+-specific coliphage were studied in three experiments using inoculants of sewage influent, sewage effluent and urban storm drain run-off. Effects of temperature, nutrients, total suspended solids, bacterial load and solar irradiation were studied in fresh and seawater matrices. Results demonstrated that temperature and solar irradiation had significant effects upon rates of inactivation (anova, P < 0.001). Inactivation rates were similar, regardless of the inoculant type. EC degraded the slowest in the dark with T90s of 115-121 and 144-177 h at 20 and 14 degrees C, respectively. When incubated in sunlight, EC was inactivated significantly more rapidly than either E. coli or F+-specific coliphage (P < 0.001). CONCLUSIONS: Inactivation of indicator bacteria is not dependent upon the original source of contamination. Inactivation rates of indicator bacteria were similar in fresh and seawater matrices. However, EC degraded more rapidly in sunlight than E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the source of faecal contamination is not an important factor to inactivation rates of indicator bacteria. However, rates of inactivation of indicator bacteria are likely system specific.     

Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.     

Effect of chlorination on the development of marine biofilms dominated by diatoms

This study addressed the antifouling efficiency of commercially available chlorine at different concentrations (0.5%, 1%, and 2%) and exposure times (0.5?min, 1?min, 5?min, and 15 min). The rapid and non-destructive FIRe (fluorescence induction and relaxation) technique was used to evaluate the effects of the biocide on diatom dominated biofilms. The efficiency of chlorine in removing diatoms from the developed biofilms increased with an increase in concentration and exposure time. The fluorescence measurements revealed low F(v)/F(m) and high σ(PSII) values for chlorine-treated Navicula and Amphora biofilms indicating that chlorination was efficient in damaging the photosystem-II reaction centers. Chlorination also caused mortality of diatom cells by damaging the cell body. In natural biofilms, the biocidal effect of chlorine was species specific; species of Amphiphrora, Navicula, Cylindrotheca, and Coscinodiscus showed an increase in the density of the population, but species of Pleurosigma, Amphora, and Thalassionema did not increase in density after chlorine treatment. It was also demonstrated that diatoms can colonize, grow and photosynthesize on chlorine-treated surfaces. Under pulse chlorination (treatment every 6 h), irrespective of chlorine concentration, the development of biofouling decreased with an increase in exposure time. Differences between exposure times of 1 to 15?min were not significant. Additionally, transmission levels of the control (non-chlorine-treated) fouled coupons reduced significantly (～20%) compared to the chlorine-treated fouled coupons (<2%). These results suggest that chlorine can be used as a biocide to control the development of diatom biofilms.     

A marine bacterial adhesion microplate test using the DAPI fluorescent dye: a new method to screen antifouling agents

AIMS: To develop a method to screen antifouling agents against marine bacterial adhesion as a sensitive, rapid and quantitative microplate fluorescent test. METHODS AND RESULTS: Our experimental method is based on a natural biofilm formed by mono-incubation of the marine bacterium Pseudoalteromonas sp. D41 in sterile natural sea water in a 96-well polystyrene microplate. The 4'6-diamidino-2-phenylindole dye was used to quantify adhered bacteria in each well. The total measured fluorescence in the wells was correlated with the amount of bacteria showing a detection limit of one bacterium per 5 microm(2) and quantifying 2 x 10(7) to 2 x 10(8) bacteria adhered per cm(2). The antifouling properties of three commercial surface-active agents and chlorine were tested by this method in the prevention of adhesion and also in the detachment of already adhered bacteria. The marine bacterial adhesion inhibition rate depending on the agent concentration showed a sigmoid shaped dose-response curve. CONCLUSIONS: This test is well adapted for a rapid and quantitative first screening of antifouling agents directly in seawater in the early steps of marine biofilm formation. Significance AND IMPACT OF THE STUDY: In contrast to the usual screenings of antifouling products which detect a bactericidal activity, this test is more appropriate to screen antifouling agents for bacterial adhesion removal or bacterial adhesion inhibition activities. This screening test focuses on the antifouling properties of the products, especially the initial steps of marine biofilm formation.     

Membrane Damage and Microbial Inactivation by Chlorine in the Absence and Presence of a Chlorine-Demanding Substrate

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.     

Glutaraldehyde: its uptake by sporing and non-sporing bacteria, rubber, plastic and an endoscope

Uptake of glutaraldehyde to bacterial spores, germinating and outgrowing spores, vegetative cells (sporing and non-sporing bacteria), various types of rubber, plastic and an endoscope was investigated. Escherichia coli NCTC 10418 exhibited greatest uptake, followed by Bacillus subtilis NCTC 8236 vegetative cells and Staphylococcus aureus NCTC 6571. Germinated and outgrowing B. subtilis spores adsorbed more glutaraldehyde than resting spores, but less than vegetative cells. Low concentrations of alkaline and acid glutaraldehyde increased the surface hydrophobicity and inhibited the germination of bacterial spores, the alkaline solution to a greater extent in both cases. Rubbers exhibited varying degrees of uptake and are listed in decreasing order of uptake: red rubber, fluorinated rubber (Vinescol), silicone rubber (Silescol), butyl rubber (Butyl XX). Polypropylene, the only plastic examined, was found not to adsorb any glutaraldehyde. The endoscope adsorbed more glutaraldehyde (per gram) than fluorinated rubber but less than red rubber. No damage was observed.     

Glutaraldehyde: its uptake by sporing and non-sporing bacteria, rubber, plastic and an endoscope

Uptake of glutaraldehyde to bacterial spores, germinating and outgrowing spores, vegetative cells (sporing and non-sporing bacteria), various types of rubber, plastic and an endoscope was investigated. Escherichia coli NCTC 10418 exhibited greatest uptake, followed by Bacillus subtilis NCTC 8236 vegetative cells and Staphylococcus aureus NCTC 6571. Germinated and outgrowing B. subtilis spores adsorbed more glutaraldehyde than resting spores, but less than vegetative cells. Low concentrations of alkaline and acid glutaraldehyde increased the surface hydrophobicity and inhibited the germination of bacterial spores, the alkaline solution to a greater extent in both cases.
Rubbers exhibited varying degrees of uptake and are listed in decreasing order of uptake: red rubber, fluorinated rubber (Vinescol), silicone rubber (Silescol), butyl rubber (Butyl XX). Polypropylene, the only plastic examined, was found not to adsorb any glutaraldehyde. The endoscope adsorbed more glutaraldehyde (per gram) than fluorinated rubber but less than red rubber. No damage was observed.     

Deep sequencing reveals exceptional diversity and modes of transmission for bacterial sponge symbionts

Marine sponges contain complex bacterial communities of considerable ecological and biotechnological importance, with many of these organisms postulated to be specific to sponge hosts. Testing this hypothesis in light of the recent discovery of the rare microbial biosphere, we investigated three Australian sponges by massively parallel 16S rRNA gene tag pyrosequencing. Here we show bacterial diversity that is unparalleled in an invertebrate host, with more than 250,000 sponge-derived sequence tags being assigned to 23 bacterial phyla and revealing up to 2996 operational taxonomic units (95% sequence similarity) per sponge species. Of the 33 previously described 'sponge-specific' clusters that were detected in this study, 48% were found exclusively in adults and larvae - implying vertical transmission of these groups. The remaining taxa, including 'Poribacteria', were also found at very low abundance among the 135,000 tags retrieved from surrounding seawater. Thus, members of the rare seawater biosphere may serve as seed organisms for widely occurring symbiont populations in sponges and their host association might have evolved much more recently than previously thought.     

Survival of Microorganisms in a Simulated Martian Environment: II. Moisture and Oxygen Requirements for Germination of Bacillus cereus and Bacillus subtilis var. niger Spores1

The effects of moisture and oxygen concentration on germination of Bacillus cereus and B. subtilis var. niger spores were investigated in a simulated Martian environment. Less moisture was required for germination than for vegetative growth of both organisms. A daily freeze-thaw cycle lowered moisture requirements for spore germination and vegetative growth of both organisms, as compared with a constant 35 C environment. Oxygen had a synergistic effect by lowing the moisture requirements for vegetative growth, and possibly germination, of both organisms. Oxygen was not required for spore germination of either organism, but was required for vegetative growth of B. subtilis and for sporulation of both organisms.     

Cytometric monitoring of growth, sporogenesis and spore cell sorting in Paenibacillus polymyxa (formerly Bacillus polymyxa)

AIMS: Formation of bacterial endospores is a basic process in Gram-positive bacteria and has implications for health, industry and the environment. Flow cytometry offers a practical alternative for the rapid detection, enumeration and characterization of bacterial endospores. METHODS AND RESULTS: Paenibacillus polymyxa was chosen for this study because its spores cause sporangium deformation and have thick walls with a star-shaped section. Sporulating populations were analysed with a particle analyser and a flow cytometer after labelling with propidium iodide and Syto-13. Flow cytometric detection of single spores was confirmed by optical and scanning electron microscopy after cell sorting. Four cell sub-populations were cytometrically detected in P. polymyxa cultures grown in liquid sporulation medium. Two sub-populations consisted of vegetative cells differing in both morphology and viability; the other two sub-populations consisted of spores differing in their viability. CONCLUSIONS: This work has shown that flow cytometry is a simple and fast method (less than 15 minutes for sample preparation and analysis) for the study of the sporulation in P. polymyxa. The use of this technique allowed both detection and quantification of sporulation inside a culture, and distinguished cells that differed in viability despite being morphologically identical under microscopic observation. SIGNIFICANCE AND IMPACT OF THE STUDY: Flow cytometry has been proved to be a valuable tool for the analysis of sporulation in P. polymyxa cultures, with the unique capacity of distinguishing between endospores and vegetative cells, and between live and dead cells, in the same analysis. An important percentage of non-viable endospores has been found in aged cultures using this method.     

Detection of free and plankton-associated Helicobacter pylori in seawater

AIMS: To detect both free and plankton-associated Helicobacter pylori in seawater samples collected on the Italian coast of the Adriatic Sea using a nested-PCR. METHODS AND RESULTS: Dissolved oxygen, pH, salinity and chlorophyll 'a' were the parameters recorded together with the characterization of zooplanktonic organisms. Plankton-associated H. pylori DNA was searched for in water samples filtered through 200 and 64 microm nylon nets whereas free bacteria were retained with the subsequent filtration through 0.22 microm pore-size membranes. Nested-PCR using primers for the glmM (ureC) gene was performed to reveal the presence of H. pylori. The DNA sequencing of amplified products confirmed the specificity of the assay. The sensitivity of the nested-PCR assay for H. pylori detection was 62 CFU per 100 ml in spiked water samples. Helicobacter pylori either free or bound to planktonic organisms was found in seven of 12 monthly samples. In particular, free bacteria were detected during the summer sampling and in November, December and March associated to planktonic cells. CONCLUSIONS: The presence of free and plankton-associated H. pylori in seawater suggests that it can be a significant reservoir and a potential route of transmission for the microorganism. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study seems to provide a promising background to define new and effective strategies for surveillance of this human pathogen.     

Relationship between inactivation kinetics of a Listeria monocytogenes suspension by chlorine and its chlorine demand

AIMS: Chlorine demand by Listeria monocytogenes cells and inactivation of L. monocytogenes by chlorine (0.6-1.0 mg l(-1)) at different temperatures (4, 20 and 30 degrees C) have been investigated in a batch reactor. METHODS AND RESULTS: Chlorine demand depended on the microbial concentration and was independent on the initial chlorine concentration and temperature. Chlorine decay was modelled by the addition of two first-order decay equations. Inactivation of L. monocytogenes by chlorine depended on the initial microbial concentration, initial chlorine concentration and temperature. A mathematical model based on a biphasic inactivation properly described survival curves of L. monocytogenes and a tertiary model was developed that satisfactorily predicted the inactivation of L. monocytogenes by different concentrations of initial chlorine at different temperatures. CONCLUSIONS: Both available chlorine decay and inactivation of L. monocytogenes by chlorine were biphasic and can be modelled by a two-term exponential model. SIGNIFICANCE AND IMPACT OF THE STUDY: The biphasic nature of survival curves of L. monocytogenes did not reflect the effect of a change of available chlorine concentration during the treatment. The microbial inactivation was caused by successive reactions that occur after the consumption of the chlorine by the bacterial cell components.     

Isolation of a dinoflagellate mitotic cyclin by functional complementation in yeast

Dinoflagellates are protists with permanently condensed chromosomes that lack histones and whose nuclear membrane remains intact during mitosis. These unusual nuclear characters have suggested that the typical cell cycle regulators might be slightly different than those in more typical eukaryotes. To test this, a cyclin has been isolated from the dinoflagellate Gonyaulax polyedra by functional complementation in cln123 mutant yeast. This GpCyc1 sequence contains two cyclin domains in its C-terminal region and a degradation box typical of mitotic cyclins. Similar to other dinoflagellate genes, GpCyc1 has a high copy number, with approximately 5000 copies found in the Gonyaulax genome. An antibody raised against the N-terminal region of the GpCYC1 reacts with a 68kDa protein on Western blots that is more abundant in cell cultures enriched for G2-phase cells than in those containing primarily G1-phase cells, indicating its cellular level follows a pattern expected for a mitotic cyclin. This is the first report of a cell cycle regulator cloned and sequenced from a dinoflagellate, and our results suggest control of the dinoflagellate cell cycle will be very similar to that of other organisms.