Effect of osmotic shock on the viability, optical density and permeability of heated Escherichia coli cells

The object of this work was to study the effect of a short incubation in 0.01 M tris buffer, pH 7.0, with a different NaCl content (0-10%) on the viability, optic density and permeability of intact and heated at 52 degrees C Escherichia coli B/r cells. In contrast to the intact cells, the viability of the heated cells depended on osmotic pressure in the medium into which they were transferred after heating. The survival rate was highest when the cells were transferred into an isotonic buffer. In the case of hypotonic and hypertonic media, the survival rate of the cells decreased owing to the death of cells which were responsible for the formation of small colonies under the isotonic conditions. This was accompanied with a more intensive drop in the optic density of bacterial suspensions while their permeability increased (when the cells were transferred into the hypotonic conditions). The role of membranes in the processes of bacterial heat inactivation is discussed on the basis of the results obtained.     

Mechanism of thermoresistance in Escherichia coli cells exposed to temperature elevation

The influence of media with different osmotic pressure (NaCl water solution) and chloramphenicol (10 micrograms/ml) on the survival, permeability, and survival curve shape of Escherichia coli B/r and E. coli Bs-1 cells, heated up to 50, 52, and 60 degrees C was investigated. As shown, the survival curve of cells heated up to 60 degrees C in isotonic conditions was characterized by exponential shape, while the survival curves of cells heated up to 50 and 52 degrees C consisted of two components characterizing thermosensitive and thermoresistant parts of cell population. Hypertonic conditions of heat at 52 degrees C decreased cell lethality and permeability. In this case, survival curves were characterized by exponential shape. Chloramphenicol was shown to protect against damaging action of heat at 50 degrees C and not to affect the viability of cells heated at 52 and 60 degrees C. It is proposed that the increase of cell thermoresistance with heat dose elevation at 50 and 52 degrees C in isotonic conditions, which is accompanied by the appearance of thermotolerant components on survival curves, may be associated with accommodational cell reactions. The essence of these reactions consists in stabilization of the osmotic cell homeostasis.     

Osmotic modification of thermal damage in Escherichia coli bacteria at various pH values of the media

A study was made of the influence of media with different osmotic pressure on cell survival and on optic density of supernatants from Escherichia coli B/r and E. coli Bs-1 cell suspensions heated under different pH values of media. Hyperthermia induced cell death accompanied with the loss of optically active (lambda = 260 nm) material. Both cell damage effects were increased in acid and alkaline conditions, compared to neutral condition of heating. Hypertonic media results in a decrease in thermic cell death and loss of cell substances. Under this condition, the protection influence of high osmotic pressure was seen to increase significantly in acid and alkaline conditions of heating, compared to neutral condition. It has been proposed that a higher thermal damage of microorganisms in acid and alkaline beating conditions and protection influence of hypertonic media, especially expressed in acid and alkaline medium, is caused to a great extent by the status of osmotic cell homeostasis.     

Features of consequences of osmotic modification at different steps of cell heating]

The influence of media with different osmotic pressure (NaCl water solutions) on survival and permeability of Escherichia coli B/r and Escherichia coli Bs-1 cells heated up to 50, 52 and 60 degrees C was investigated. Hypotonic media increased, while hypertonic media, within a certain range of sodium chloride concentrations, decreased the damaging action of heating independently of the temperature. The effectiveness of thermoprotection was seen to increase, and the range of osmolyte concentrations, at which the highest effect of protection takes place, to move markedly towards higher concentrations of NaCl with increase in heating temperature. A certain relationship is suggested between the observed phenomenon and the osmotic homeostasis system of microorganisms under condition of thermogenic and tonic stress.     

About the characters of thermoprotection in influence of osmolites on bacteria

The influence of the length of staying of Escherichia coli B/r cells in hypertonic NaCl solution before heating at 52 and 60 degrees C on the magnitude of salt thermoprotection was investigated. In addition, the dependence of the isotonic and thermoprotective NaCl concentrations on the exposure temperature was investigated. It was shown that the volume of cell osmotic thermoprotection was independent on the length of preliminary staying of microorganisms in hypertonic NaCl solution. It was also shown that the magnitude of isotonic and thermoprotective osmolite concentrations increased with the increase in the exposure temperature. The analysis of the data obtained and published in literature indicates that the compensating mechanism is involved in salt bacteria thermoprotection rather than the dehydratation one.     

渗透压对痢疾志贺菌水通道蛋白glpF基因表达的影响

目的探讨细菌水-甘油通道蛋白（GlpF）的生理功能及其对生长繁殖的影响。方法将用简并PCR发现表达水通道蛋白glpF基因的痢疾志贺菌接种于不同渗透压的液体培养基和添加了GlpF功能抑制剂的相应培养基中,培养不同时间后取培养液检测其生长繁殖量,RT-PCR分析其GlpF的表达。结果痢疾志贺菌GlpF的表达随培养基渗透压的改变而变化,在低渗培养基中的表达低于在等渗环境中;在高渗透压的培养基中,其表达显著高于在等渗培养基中。在加入Hg2＋抑制剂抑制GlpF的表达后,在低渗培养基中,未明显影响细菌的生长繁殖,但在较高渗透压的培养基中,细菌的繁殖量显著少于在未加Hg2＋抑制剂的同样渗透压培养基中。结论在非等渗环境中,细菌GlpF的表达对细菌细胞内外水分的调节,维持胞内环境稳定起到重要作用,尤其在高渗透压环境中更为明显。     

The influence of cyclic heating and cooling on Escherichia coli survival

Repeated heating and cooling in lethal (2-52 degrees C) and nonlethal (2-37 degrees C) temperature ranges resulted in cell death of Escherichia coli B/r and E. coli B(S-1) suspended in 0.01 M phosphate buffer, pH 7.0 at varying osmotic pressure, but not in cow's milk. The lethal effect increased with the rate of heating and with increasing suspension media tonicity; it may be caused by the temperature destabilization of cellular osmotic homeostasis.     

渗透压突然改变对大肠埃希菌水通道蛋白GlpF基因表达的影响

目的 探讨水-甘油通道蛋白(glycerol protein ficilitator,GlpF)的生理功能及对细菌生长繁殖的影响.方法 将大肠埃希菌接种于等渗透压的液体培养基(1 IM)培养,18h后,突然改变培养液的渗透压,在30、60、120 min时间点检测细菌的A600nm值,RT-PCR分析其GlpF的表达.结果 突然改变渗透压后,大肠埃希菌数量均有不同程度的减少.在1/2 IM组、1/4 IM组细菌的A600nm值与其各自对照组相比,变化并不明显,但其细菌GlpF表达量明显降低.在高渗组,2 IM组的A600nm值与等渗组相比变化不大,而其GlpF表达量明显高于等渗组.结论 在环境渗透压突然改变时,细菌可以通过调控GlpF的表达来实现对细菌细胞内外水份的调节,以维持胞内环境稳定.     

Thermal resistance of intracellular Listeria monocytogenes cells suspended in raw bovine milk.

The thermal resistance of Listeria monocytogenes associated with a milk-borne outbreak of listeriosis was determined in parallel experiments by using freely suspended bacteria and bacteria internalized by phagocytes. The latter inoculum was generated by an in vitro phagocytosis reaction with immune-antigen-elicited murine peritoneal phagocytes. The heat suspension medium was raw whole bovine milk. Both suspensions were heated at temperatures ranging from 52.2 to 71.7 degrees C for various periods of time. Mean D values for each temperature and condition of heated suspension revealed no significant differences. The extrapolated D71.7 degrees C (161 degrees F) value for bacteria internalized by phagocytes was 1.9 s. Combined tube and slug-flow heat exchanger results yielded an estimated D71.7 degrees C value of 1.6 s for freely suspended bacteria. The intracellular position did not protect L. monocytogenes from thermal inactivation.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes.

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating

Storage of cultures of Salmonella enteritidis PT4 at either 4 degrees or 8 degrees C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4-7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60 degrees rather than 55 degrees C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37 degrees C prior to heating enabled the organisms to recover heat resistance.     

Plasma hyperosmolality augments peripheral vascular response to baroreceptor unloading during heat stress

The aim of this study was to elucidate the interactive effect of central hypovolemia and plasma hyperosmolality on regulation of peripheral vascular response and AVP secretion during heat stress. Seven male subjects were infused with either isotonic (0.9%; NOSM) or hypertonic (3.0%; HOSM) NaCl solution and then heated by perfusing 42 degrees C (heat stress; HT) or 34.5 degrees C water (normothermia; NT) through water perfusion suits. Sixty minutes later, subjects were exposed to progressive lower body negative pressure (LBNP) to -40 mmHg. Plasma osmolality (P(osmol)) increased by approximately 11 mosmol/kgH(2)O in HOSM conditions. The increase in esophageal temperature before LBNP was much larger in HT-HOSM (0.90 +/- 0.09 degrees C) than in HT-NOSM (0.30 +/- 0.07 degrees C) (P < 0.01) because of osmotic inhibition of thermoregulation. During LBNP, mean arterial pressure was well maintained, and changes in thoracic impedance and stroke volume were similar in all conditions. Forearm vascular conductance (FVC) before application of LBNP was higher in HT than in NT conditions (P < 0.001) and was not influenced by P(osmol) within the thermal conditions. The reduction in FVC at -40 mmHg in HT-HOSM (-9.99 +/- 0.96 units; 58.8 +/- 4.1%) was significantly larger than in HT-NOSM (-6.02 +/- 1.23 units; 44.7 +/- 8.1%) (P < 0.05), whereas the FVC response was not different between NT-NOSM and NT-HOSM. Plasma AVP response to LBNP did not interact with P(osmol) in either NT or HT conditions. These data indicate that there apparently exists an interactive effect of P(osmol) and central hypovolemia on the peripheral vascular response during heat stress, or peripheral vasodilated conditions, but not in normothermia.     

Cold and hyperosmolar fluids in canine trachea: vascular and smooth muscle tone and albumin flux

We have studied the effects of liquids of various osmolalities and temperatures on the tracheal vasculature, smooth muscle tone, and transepithelial albumin flux. In 10 anesthetized dogs a 10- to 13-cm length of cervical trachea was cannulated to allow instillation of fluids into its lumen. The cranial tracheal arteries were perfused at constant flow, with monitoring of the perfusion pressures (Ptr) and the external tracheal diameter (Dtr). Control fluid was Krebs-Henseleit solution (KH) with NaCl added to result in a 325-mosM solution (isotonic). Hypertonic solutions were KH with NaCl (warm hypertonic) or glucose (hypertonic glucose) added to result in a 800-mosM solution. All solutions were at 38 degrees C, with isotonic and the hypertonic NaCl solutions also given at 18 degrees C (cold isotonic and cold hypertonic). Fluorescent labeled albumin was given intravenously, and the change in fluorescence in the fluid was measured during each 15-min period. Changing from warm isotonic to cold isotonic decreased Dtr and Ptr. Changing from warm isotonic to warm hypertonic or hypertonic glucose decreased Ptr with no change in Dtr. The cold hypertonic responses were not different from cold isotonic responses. Warm hypertonic solution increased albumin flux into the tracheal lumen over a 15-min period to three times that of the control period, persisting for 15 min after replacement with warm isotonic solution. Cooling induces a vasodilation and smooth muscle contraction of the trachea, whereas hypertonic solutions result in vasodilation and, if osmolality is increased with NaCl, an increase in albumin flux into the tracheal lumen.     

Volume changes of an endothelial cell monolayer on exposure to anisotonic media

The osmotic process plays an important role in controlling the distribution of water across cell membranes and thus the cell volume. A system was designed to detect the volume changes of an endothelial cell monolayer when cells were exposed to media with altered osmolalities. Electrodes housed in a flow chamber measured the resistance of ionic media flowing over a cultured cell layer. Assuming the cell membrane acts as an electrical insulator, volume changes of the cell layer can be calculated from the corresponding changes in chamber resistance. The media used in the experiments had osmolalities in the range 120-630 mmol/kg. When cells were exposed to hypertonic media, there was rapid shrinkage with an approximate 30% reduction in total cell volume for a twofold increase in osmolality. On exposure to hypotonic media, the cells initially swelled with an approximate 20% volume increase for a decrease in osmolality by half. With sustained exposure to low osmolality media, there was a gradual and partial return of cell volume towards isotonic values that started 10 minutes after and was complete within 30 minutes of the osmolality alteration. This finding suggests regulatory volume decrease (RVD); however, no regulatory volume increase (RVI) was observed with the continued exposure to hypertonic media over 45 minutes.     

The influence of cooling rates and medium tonicity on Escherichia coli B/r survival after heating with different lethal temperatures

Cell thermosensitivity of Escherichia coli B/r increases with the cooling rise, especially in hypertonic conditions after heating at 50, 55 and 60 degrees C. A certain relationship is suggested between the observed phenomenon and the osmotic homeostasis system of microorganisms under condition of thermogenic and tonic stress.     

The influenza virus-induced fusion of erythrocyte ghosts does not depend on osmotic forces

The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.     

The Mechanics of Injury to Isolated Protoplasts following Osmotic Contraction and Expansion

Micro-osmotic manipulation was used to determine the influence of osmotic contraction on the expansion potential of individual protoplasts isolated from rye (Secale cereale L. cv Puma) leaves. For protoplasts isolated from leaves of nonacclimated plants (NA protoplasts), osmotic contraction in sufficiently hypertonic solutions (>1.53 osmolal) predisposed the protoplasts to lysis during osmotic expansion when they were returned to isotonic conditions (0.53 osmolal). In contrast, for protoplasts isolated from leaves of cold acclimated plants (ACC protoplasts), osmotic contraction in either 2.6 or 4.0 osmolal solutions was readily reversible. Following osmotic contraction, the resting tension (γ_r) of NA protoplasts was similar to that determined for protoplasts in isotonic solutions (i.e. 110 ± 22 micronewtons per meter). In contrast, γ_r of ACC protoplasts decreased from 164 ± 27 micronewtons per meter in isotonic solutions to values close to zero in hypertonic solutions. Following expansion in hypotonic solutions, γ_r's of both NA and ACC protoplasts were similar for area expansions over the range of 1.3 to 1.6. Following osmotic contraction and reexpansion of NA protoplasts, hysteresis was observed in the relationship between γ_r and surface area—with higher values of γ_r at a given surface area. In contrast, no hysteresis was observed in this relationship for ACC protoplasts. Direct measurements of plasma membrane tension (γ) during osmotic expansion of NA protoplasts from hypertonic solutions (1.53 osmolal) revealed that γ increased rapidly after small increments in surface area, and lysis occurred over a range of 1.2 to 8 millinewtons per meter. During osmotic expansion of ACC protoplasts from hypertonic solutions (2.6 osmolal), there was little increase in γ until after the isotonic surface area was exceeded. These results are discussed in relation to the differences in the behavior of the plasma membrane of NA and ACC protoplasts during osmotic contraction (i.e. endocytotic vesiculation versus exocytotic extrusion) and provide a mechanistic interpretation to account for the differential sensitivity of NA and ACC protoplasts to osmotic expansion from hypertonic solutions.     

Destruction and injury of Escherichia coli during microwave heating under vacuum

AIMS: To study the effect of 2450 MHz microwave radiation under vacuum (vacuum microwave or VM) on survival and injury of Escherichia coli and to search for possible nonthermal effects associated with VM. METHODS AND RESULTS: Destruction kinetics of E. coli in peptone water were determined in a continuous-flow vacuum system, heated by convection heating in a water bath or with microwaves (VMs). Vacuum was used to control the boiling point of water and to maintain temperature in the bacterial suspensions at specified levels (49-64 degrees C). CONCLUSIONS: z-Value in the water bath treatment was 9.1 degrees C while for VM at 510 and 711 W it was 6.2 and 5.9 degrees C, suggesting that E. coli is more sensitive to temperature changes under microwave heating. Arrhenius calculations of the activation energies of the destruction reactions suggest that the mechanism of destruction in VM may be different from that of conventional heat. The number of injured micro-organisms showed no significant differences among treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of temperature on E. coli destruction was different when microwaves were the medium of heat transfer, suggesting the existence of factors other than heat contributing to the lethal effect of VM.     

Effect of hydration on the water content of human erythrocytes.

An ideal, hydrated, nondilute pseudobinary salt-protein-water solution model of the RBC intracellular solution has been developed to describe the osmotic behavior of human erythrocytes during freezing and thawing. Because of the hydration of intracellular solutes (mostly cell proteins), our analytical results predict that at least 16.65% of the isotonic cell water content will be retained within RBCs placed in hypertonic solutions. These findings are consistent not only with the experimental measurements of the amount of isotonic cell water retained within RBCs subjected to nonisotonic extracellular solutions (20-32%) but also with the experimental evidence that all of the water within RBCs is solvent water. By modeling the RBC intracellular solution as a hydrated salt-protein-water solution, no anomalous osmotic behavior is apparent.