Vibrio anguillarum: influence of temperature, pH, NaCl concentration and incubation time on growth

An analysis of the effect of growth conditions on the growth (O.D. values) of five Vibrio anguillarum strains showed that the optima were as follows: pH 7, temperature 25 degrees C, NaCl concentration 2%, and O.D. estimates increased with the incubation time. The independent parameters, as well as their interactions significantly influenced the growth of Vibrio anguillarum (P less than 0.0001). Only the strain-salinity interaction was not always statistically significant. A restriction of the parameters to a level relevant for Danish marine recipients showed that pH and NaCl concentration (salinity) might be of minor importance while the temperature was always of high significance. The possible impact of these observations on local conditions is discussed.     

The effect of incubation temperature and sodium chloride concentration on the growth kinetics of Vibrio anguillarum and Vibrio anguillarum-related organisms

The effect of temperature and NaCl concentration on the growth kinetics of Vibrio anguillarum and V. anguillarum -related (VAR) strains was studied.
For one wild VAR strain, NaCl concentration interfered with growth temperature parameters, in particular, with the maximum growth temperature but also with the optimum temperature (defined as the temperature at which μ_max equals its maximal value μ_opt), and with μ_opt itself. For the same strain, optimal growth required the adding of NaCl to the medium to a final concentration of 1·5%. These results were not confirmed by tests on a V. anguillarum collection strain.
When the NaCl concentration in the culture media was 1.5%, the optmum temperature for the nine strains studied ranged from 29.7°C to 34°C whereas the maximum temperature ranged between 35.3°C and 38.5°C.
Hence, antbiotic susceptibility testing as well as biochemical identification might be carried out at 30°C in the presence of 1.5% NaCl, which corresponded to a suboptimal growth.     

Influence of hematoporphyrin derivative concentration, incubation time, temperature during incubation and laser dose fractionation on photosensitivity of normal hemopoietic progenitors or leukemic cells

Photodynamic therapy represents a new approach for the local control of cancers. It has recently been claimed that photodynamic therapy mediated by hematoporphyrin derivative (HPD) is selectively more efficient for killing leukemic cells than normal progenitors. To improve this effect, we studied the influence of hematoporphyrin dose, temperature during incubation and/or treatment, hematoporphyrin derivative incubation time, and fractionation of the argon laser light (488-514 nm) used for hematoporphyrin stimulation. Plating efficiency calculated after a 7-day period of growth on collagen gel medium showed a dose-dependent phototoxicity of HPD reaching 0.01% for normal hemopoietic progenitors and 0.001% for leukemic cells (dose = 12.5 micrograms/ml). The 10:1 ratio of normal hemopoietic progenitors to leukemic cells was also found to be the same or increased when temperature was 37 degrees C during incubation and 4 degrees C during laser irradiation. Similar results were also found when incubation time was varied from 75-120 min, or when laser irradiation dose was fractionated into 2 or 3 periods. The ratio of normal progenitors to leukemic cells reached 100:1 when 75 J/cm2 were fractionated into 3 periods after an incubation time of 120 min with 10 micrograms/ml HPD. Selectivity in photodynamic treatment seems to occur between normal hemopoietic progenitors and leukemic cells. The mechanism of this selectivity remains unclear, but experiments with the fractionated irradiation dose suggest that as in radiotherapy, better potentially lethal damage repair in normal cells could be a factor for selectivity in photodynamic therapy. Our results obtained with leukemic cells are fully in agreement with data in the literature concerning similar experimental models.     

Molecular diffusion into ferritin: pathways, temperature dependence, incubation time, and concentration effects

The detailed kinetics of permeation and effusion of small nitroxide spin probe radicals with the protein shells of horse spleen ferritin (HoSF) and human H-chain ferritin (HuHF) and a 3-fold channel variant D131H+E134H of HuHF were studied by electron paramagnetic resonance spectroscopy and gel permeation chromatography under a variety of experimental conditions. The results confirm that the permeation of molecular species of 7-9-A diameter into ferritin is a charge selective process and that the threefold channels are the likely pathways for entry into the protein. Studies with holoHoSF show that increased temperature increases the rates of penetration and effusion and also increases the concentration of positively charged spin probe accumulated within the protein in excess of that in the external solution. The interior of HoSF is much more accessible to small molecules at physiological temperature of approximately 40 degrees C than at room temperature. The large activation energy of 63-67 kJ/mol measured for the effusion/penetration and the small diffusion coefficient, D approximately 5 x 10(-22) m(2)/s at 20 degrees C, corresponding to a time of approximately 60 min for traversing the protein shell, is consistent with the kinetics of diffusion being largely controlled by the restrictive porosity of the protein itself. An inverse dependence of the first-order rate constant for effusion out of the protein channel on the incubation time used for radical penetration into the protein is attributed to increased binding of the radical within the funnel-shaped channel.     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia.

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of NaCl, pH, temperature, and atmosphere on growth of Salmonella typhimurium in glucose-mineral salts medium

The interactions of pH (5.0, 6.0, and 7.0), temperature (19, 28, and 37 degrees C), and atmosphere (aerobic versus anaerobic) with NaCl (0, 1, 2, 3, 4, and 5%) on the growth of Salmonella typhimurium ATCC 14028 in defined glucose-mineral salts culture medium were evaluated. Response surface methodology was used to develop equations describing the response of S. typhimurium to environmental changes. The response to an increasing concentration of NaCl at any temperature tested was nonlinear. The maximum growth was predicted to occur at an NaCl concentration of 0.5%, a temperature of 19 degrees C, and an initial pH of 7.0 under aerobic growth conditions. The relative amounts of aerobic growth at 19 degrees C, pH 7.0, and NaCl concentrations of 0, 0.5, 1, 2, 3, 4, and 5% were predicted to be 99.2, 100.0, 98.8, 90.2, 73.5, 48.6, and 15.6%, respectively. Anaerobic growth conditions repressed the amount of growth relative to that under aerobic conditions, and the effects of NaCl and pH were additive at low salt concentrations; however, at higher salt levels anaerobiosis provided protection against the effects of NaCl.     

Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration

The conditions that optimize Sendai virus membrane fusion with liposomes have been studied. No fusion occurs in the absence of ganglioside receptors. Maximum fusion occurs when the molar ratio of ganglioside GD1a to phospholipid is 0.02 or greater. The amount of fusion at 37 degrees C increases with time up to at least 6.5 h. The rate of fusion increases from the lowest temperature tested, 10 degrees C, to 40 degrees C. Above 43 degrees C the amount of fusion decreases because of thermal inactivation of the viral proteins. There is a broad pH maximum between pH 7.5 and pH 9.0. At both ends of the pH range the amount of fusion increases and exceeds that found in the physiologic pH range. Neither ethylenediaminetetraacetic acid nor Ca2+ changes the amount of membrane fusion. The optimal conditions for membrane fusion of Sendai virus membranes with liposomes are the same as the optimal conditions for fusion with host cells and with red blood cells. Since the liposomes contain no proteins, the optimal conditions for Sendai virus membrane fusion must be determined by the viral proteins and be mostly independent of the nature or presence of the host proteins.     

Effect of NaCl, pH, temperature, and atmosphere on growth of Salmonella typhimurium in glucose-mineral salts medium.

The interactions of pH (5.0, 6.0, and 7.0), temperature (19, 28, and 37 degrees C), and atmosphere (aerobic versus anaerobic) with NaCl (0, 1, 2, 3, 4, and 5%) on the growth of Salmonella typhimurium ATCC 14028 in defined glucose-mineral salts culture medium were evaluated. Response surface methodology was used to develop equations describing the response of S. typhimurium to environmental changes. The response to an increasing concentration of NaCl at any temperature tested was nonlinear. The maximum growth was predicted to occur at an NaCl concentration of 0.5%, a temperature of 19 degrees C, and an initial pH of 7.0 under aerobic growth conditions. The relative amounts of aerobic growth at 19 degrees C, pH 7.0, and NaCl concentrations of 0, 0.5, 1, 2, 3, 4, and 5% were predicted to be 99.2, 100.0, 98.8, 90.2, 73.5, 48.6, and 15.6%, respectively. Anaerobic growth conditions repressed the amount of growth relative to that under aerobic conditions, and the effects of NaCl and pH were additive at low salt concentrations; however, at higher salt levels anaerobiosis provided protection against the effects of NaCl.     

Profiling of acylated homoserine lactones of Vibrio anguillarum in vitro and in vivo: Influence of growth conditions and serotype

Vibrio anguillarum produces several interlinked acylated homoserine lactone (AHL) signal molecules which may influence expression of its virulence factors such as exoprotease production and biofilm formation. Using both thin layer chromatography and HPLC-high resolution mass spectrometry (HPLC-HRMS), we demonstrate in this study that the same types of AHLs are produced by many serotypes of V. anguillarum and that altering in vitro growth conditions (salinity, temperature and iron concentration) has little influence on the AHL-profile. Most strains produced N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) and N-(3-hydroxy-hexanoyl)-l-homoserine lactone (3-hydroxy-C6-HSL) as the dominant molecules. Also, two spots with AHL activity appeared on TLC plates, which could not be identified as AHL structures. Trace amounts of N-(3-hydroxy-octanoyl)-l-homoserine lactone, N-(3-hydroxy-decanoyl)-l-homoserine lactone and N-(3-hydroxy-dodecanoyl)-l-homoserine lactone (3-hydroxy-C8-HSL, 3-hydroxy-C10-HSL and 3-oxo-C12-HSL, respectively) were also detected by HPLC-HRMS analysis from in vitro cultures. Most studies of quorum sensing (QS) systems have been conducted in vitro, the purpose of our study was to determine if the same acylated homoserine lactones were produced in vivo during infection. Extracts from infected fish were purified using several solid phase extraction strategies to allow chromatographic detection and separation by both TLC and HLPC-HRMS. 3-oxo-C10-HSL and 3-hydroxy-C6-HSL were detected in organs from fish dying from vibriosis, however, compared to in vitro culturing where 3-oxo-C10-HSL is the dominant molecule, 3-hydroxy-C6-HSL was prominent in the infected fish tissues. Hence, the balance between the QS systems may be different during infection compared to in vitro cultures. For future studies of QS systems and the possible specific interference with expression of virulence factors, in vitro cultures should be optimised to reflect the in vivo situation.     

Influence of setting-up time, temperature of incubation and age on the transmural potential difference across chicken rectum

The effects of different experimental conditions on the in vitro transmural potential difference (PD) have been studied in the chicken rectum by the Ussing and Zerahn technique. Results have been analyzed with a statistical method to reveal the contribution of different controlled variables to the response, as well as the possible contribution of interactions between them. The variables considered were: age, setting-up time, temperature of the incubation medium and time elapsed from the beginning of incubation. It can be concluded that a) PD increases when temperature rises from 31 degrees C to 37 degrees C and when age increases from 71 to 123 days; b) Changes in PD during incubation depend on the temperature of incubation medium; c) The influence of age on PD depends on the temperature of the incubation medium and on the time required to set-up the preparation; d) PD is inversely co-related with setting-up times ranging from 3.5 to 7.5 min.     

Relevance of incubation temperature for Vibrio salmonicida vaccine production

AIMS: To investigate the relationships between water temperature, bacterial growth, virulence and antigen expression in Vibrio salmonicida, the causal agent of cold water vibriosis in Atlantic salmon (Salmo salar L.). METHODS AND RESULTS: The significance of sea temperature was investigated using historical clinical and oceanographic data. An upper threshold for disease of approx. 10 degrees C was established. The effects of culture temperature and media type on bacterial growth were studied on solid and in liquid media. The highest rates of cell division were identified at 15 degrees C on solid media and 10 degrees C in liquid media. Outer membrane protein (OMP) expression and serological response in Atlantic salmon were studied using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. A novel 76-kDa OMP produced in unshaken cultures at 10 degrees C was not found to stimulate a specific humoral response. CONCLUSIONS: Diagnostic agar plate-based incubation of suspected V. salmonicida should be carried out at 15 degrees C. High yield broth cultures for vaccine production should be incubated at 10 degrees C or lower. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is, to the best of our knowledge, the first to identify different optimal temperatures in a bacterial species cultured on physically different types of media. The evidence presented suggests that V. salmonicida and possibly other bacteria destined for vaccine use in poikilothermic organisms should be cultured at temperatures consistent with that at which disease occurs.     

The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10(4) cells were 4.66 at 30 degrees C, 4.36 at 10 degrees C and 4.19 at 5 degrees C. These limits were salt dependent, low (4-6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4.66 at 30 degrees C but this was increased to 4.83 at 10 degrees C. At 5 degrees C growth occurred at pH 7.0 but not at pH 5.13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10 degrees C and 30 degrees C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10⁴ cells were 4·66 at 30†C, 4·36 at 10†C and 4·19 at 5†C. These limits were salt dependent, low (4–6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4·66 at 30†C but this was increased to 4·83 at 10†C. At 5†C growth occurred at pH 7·0 but not at pH 5·13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10†C and 30†C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.