The effect of incubation time and temperature on growth of Escherichia coli on gradient plates containing sodium chloride and sodium nitrite

Two-dimensional gradient plates are a convenient way of screening antimicrobial effects of preservative factors acting in combination across a broad range of physical and chemical conditions. We report the effects of sodium chloride, sodium nitrite and incubation temperature on the growth of Escherichia coli by staining, laser densitometry and computer graphics. Staining not only more easily distinguished the growth area but also gave an indication of the viability of cells present. 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride was the more useful of the two stains used. Inhibitory concentrations of sodium chloride decreased with reduced incubation temperature. The response of E. coli to combinations of salt and nitrite on gradient plates was very similar to its response in liquid medium.     

Comparison of the effects of sodium chloride, pH and temperature on the growth of Listeria monocytogenes on gradient plates and in liquid medium

Gradient plates provide a convenient means of assessing the effect of sodium chloride and pH in combination with different temperatures on the growth of microorganisms. The effects of these variables on the growth of Listeria monocytogenes in gradient plates at four temperatures are compared with growth in a micro-well system. The growth-limiting conditions in the two systems are in good agreement, and also compare favourably with data in the literature. The gradient plates gave a good illustration of the concentrations of sodium chloride required to inhibit growth at different temperatures and pHs, although incubation time is limited to 72 h.     

Detection of growth of Escherichia coli on two-dimensional diffusion gradient plates

One drawback of using two-dimensional diffusion gradient plates, the subjective visual assessment of microbial growth, has been overcome. Growth of Escherichia coli was detected with pH indicators in the medium or by staining growth with a biochemical stain, L-alanine- p -nitroanilide, or a respiratory dye, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride. Stained growth was scanned with a laser densitometer and traces combined in a computer to give a three-dimensional semi-quantitative representation of growth over the gradient plate.     

Expanded response surface model for predicting the effects of temperatures, pH, sodium chloride contents and sodium nitrite concentrations on the growth rate of Yersinia enterocolitica

The previously reported data set for the low temperature (5, 12 and 19°C) of Yersinia enterocolitica was expanded to include higher abusive temperature (28, 37 and 42°C). In addition to temperature, the data set included the effects and interactions of pH (4.5–8.5), sodium chloride (0.5-5%) and sodium nitrite (0-200 μg ml^-1) on the aerobic growth of Y. enterocolitica in brain heart infusion broth. Growth curves were modeled by fitting viable count data to the Gompertz equation. Quadratic models of natural logarithm transformations of the Gompertz B and M values and the derived values for lag phase durations and generation times were obtained using response surface analyses. Predictions based on the models for B and M values were comparable to predictions based on the derived values. These revised models provide an expanded means for rapidly estimating how the bacterium is likely to respond to any combination of the four variables within the specified ranges.     

Anti-microbial action against verotoxigenic Escherichia coli O157:H7 of nitric oxide derived from sodium nitrite

The levels of verotoxin-1 and verotoxin-2 released by verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were evaluated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2, and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6000 mg/l, minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with a treatment by sodium chloride or antibiotics. When the electron paramagnetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77 K to clarify the mechanism for the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appears that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated from the reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. The amount of ATP was decreased by the presence of sodium nitrite in the cell suspension. These findings indicate that nitric oxide derived from sodium nitrite penetrated the cells and inactivated enzymes related to the respiratory chain.     

Regulation of biosynthesis of l-amino acid oxidase by Neurospora crassa

A temperature gradient device has been used to produce pH-temperature gradient plates. The response of Salmonella typhimurium and Bacillus cereus to continuous gradients of pH and temperature has been mapped. A third variable, sodium chloride, was investigated by incorporating it at different concentrations in a series of gradient plates. Both organisms exhibited an optimum temperature and pH range for maximum salt tolerance.     

Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrite, polyphosphate and various sugars

The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture.     

High dietary sodium chloride and body temperature in the domestic fowl and the Glaucous-winged gull

Arad and Skadhauge (1986) correlated plasma sodium to calcium ratio and body temperature in domestic fowl (Gallus domesticus) during increased dietary sodium chloride intake which increased plasma sodium concentration. During acclimation to high dietary NaCl, body temperature should increase in proportion to the increase in plasma sodium concentration, and body temperature should increase less in gulls than in chickens because salt gland secretion of NaCl by gulls should prevent elevation of plasma sodium concentration. Plasma osmolality, plasma sodium concentration, plasma concentrations of total calcium and ionized calcium, and body temperature and panting threshold were measured in domestic roosters and Glaucous-winged gulls before and after exposure to high NaCl diets. Gull body temperature (40.4±0.2 °C) increased significantly (PM0.05) during salt acclimation. Rooster body temperature (41.0±0.2 °C) did not increase significantly. Plasma sodium concentration increased in gulls (5.4±0.5%, P<0.01) and was correlated with body temperature (r=0.497, P<0.05); the 3.8±1.0% increase in plasma sodium concentration in roosters (P<0.01) was not, suggesting that change in body temperature might be a response to the magnitude of increase in plasma sodium concentration. Plasma ionized calcium concentration increased by 12.9±4.6% (P<0.01) in gulls and by 5.3±1.0% (P<0.01) in roosters. Plasma sodium concentration was correlated with calcium ion concentration in both gulls (r=0.635, P<0.05) and roosters (r=0.664, P<0.05). In neither species were ratios of sodium to total calcium plasma concentration or sodium to ionized calcium concentration altered or related to body temperature. Panting threshold increased significantly in roosters following salt acclimation, but not in gulls due to high variability in response. The increase in gull plasma sodium concentration was small compared to previously reported (Saxena 1976; Denbow and Edens 1980, 1981; Maki et al. 1988) increases in hypothalamic and intraventricular sodium concentration following infusion of Na⁺, yet the effect on body temperature was similar in both types of studies. This suggests that sodium may have peripheral effects that augment the central effects imposed by altered hypothalamic interstitial sodium and calcium concentration.Abbreviations [Ca]_p1 total calcium concentration in plasma - [Ca²⁺] ionized calcium concentration in plasma - [Cl]_p1 chloride concentration in plasma - f respiratory frequency - Hct hematocrit - [K]_p1 potassium concentration in plasma - [Na]_p1 sodium concentration in plasma - osm_p1 plasma osmolality - PT panting threshold - T _a ambient temperature - T _b body temperature - V _t tidal volume     

Sodium chloride enhances recovery and growth of acid-stressed E. coli O157:H7

AIMS: Combinations of sodium chloride and acid are frequently used to inhibit growth of spoilage and pathogenic bacteria in food. The influence of differing sodium chloride, lactate and pH values on the growth of stressed and unstressed cells of a non-toxigenic strain of Escherichia coli O157:H7 was studied. METHODS AND RESULTS: At pH 5.5 or 6.0, there was little or no effect on the growth rate in the presence of lactate and/or sodium chloride, but the lag times were longer as the lactate concentration increased. At pH 5.0, in the absence of sodium chloride, increasing the lactate concentration increased the growth rate and the lag time; no growth occurred in the presence of 1.5 g 100 g(-1) lactate. In the presence of 4-6 g 100 g(-1) sodium chloride, growth occurred at 1.5 g 100 g(-1) lactate. The growth rate was similar at all lactate concentrations. CONCLUSION: The results demonstrate that the presence of sodium chloride promoted growth of E. coli O157:H7, especially under stressful conditions of low pH. Significance and Impact of the Study: These findings could have implications for the use of acid and sodium chloride as a preservation treatment for the inhibition of E. coli O157:H7 in food.     

Use of gradient plates to study combined effects of temperature, pH, and NaCl concentration on growth of Monascus ruber van Tieghem, an Ascomycetes fungus isolated from green table olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35 degrees C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30 degrees C but not at 35 degrees C. Growth was better at 30 and 25 degrees C as judged by the larger extent of the plates covered by mycelium compared with that at 35 degrees C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35 degrees C, whereas at 35 degrees C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

Sodium nitrite and sorbic acid effects on Clostridium botulinum spore germination and total microbial growth in chicken frankfurter emulsions during temperature abuse.

Samples of (i) a control or of (ii) sodium nitrite-containing or (iii) sorbic acid-containing, mechanically deboned chicken meat frankfurter-type emulsions inoculated with Clostridium botulinum spores, or a combination of ii and iii, were temperature abuse at 27 degrees C. Spore germination and total microbial growth were followed and examined at specified times and until toxic samples were detected. The spores germinated within 3 days in both control and nitrite (20, 40 and 156 micrograms/g) treatments. Sorbic acid (0.2%) alone or in combination with nitrite (20, 40, and 156 micrograms/g) significantly (P less than 0.05) inhibited spore germinations. No significant germination was recorded until toxic samples were detected. A much longer incubation period was necessary for toxin to be formed in nitrite-sorbic acid combination treatments as contrasted with controls or nitrite and sorbic acid used individually. Total growth was not affected by the presence of nitrite, whereas sorbic acid appeared to depress it. Possible mechanisms explaining the effects of nitrite and sorbic acid on spore germination and growth are postulated.     

Sodium nitrite and sorbic acid effects on Clostridium botulinum spore germination and total microbial growth in chicken frankfurter emulsions during temperature abuse.

Samples of (i) a control or of (ii) sodium nitrite-containing or (iii) sorbic acid-containing, mechanically deboned chicken meat frankfurter-type emulsions inoculated with Clostridium botulinum spores, or a combination of ii and iii, were temperature abuse at 27 degrees C. Spore germination and total microbial growth were followed and examined at specified times and until toxic samples were detected. The spores germinated within 3 days in both control and nitrite (20, 40 and 156 micrograms/g) treatments. Sorbic acid (0.2%) alone or in combination with nitrite (20, 40, and 156 micrograms/g) significantly (P less than 0.05) inhibited spore germinations. No significant germination was recorded until toxic samples were detected. A much longer incubation period was necessary for toxin to be formed in nitrite-sorbic acid combination treatments as contrasted with controls or nitrite and sorbic acid used individually. Total growth was not affected by the presence of nitrite, whereas sorbic acid appeared to depress it. Possible mechanisms explaining the effects of nitrite and sorbic acid on spore germination and growth are postulated.     

Effect of sodium chloride and pH on enterotoxin B production

Genigeorgis, Constantin (University of California, Davis), and Walter W. Sadler. Effect of sodium chloride and pH on enterotoxin B production. J. Bacteriol. 92:1383-1387. 1966.-The growth and production of enterotoxin B by Staphylococcus aureus strain S-6 in Brain Heart Infusion broth with 2 to 16% sodium chloride and an initial pH of 5.1 to 6.9 was studied during a 10-day incubation period at 37 C. Growth was good at pH 6.9 and with a 16% concentration of salt, but no cells survived after 10 days of incubation at pH 5.1 and with a 16% concentration of salt. With geldiffusion technique, enterotoxin B was detected in broth with pH 6.9 and up to 10% salt or pH 5.1 and up to 4% salt. Growth and enterotoxin production were better when pH was increased and salt concentration was decreased. The dependence of toxin production on the interaction of these two factors was demonstrated.     

Inhibition of growth of E. coli cells by anolites of sodium and potassium chloride after processing solutions in a diaphragmatic electrolyzer

The relationship between the inhibitory effect of sodium chloride and potassium anolites, obtained in a diaphragm electrolyser, and the physicochemical parameters of solutions was compared with that between the inhibitory effect and physicochemical properties of hypochlorites obtained after treating the solutions in an electrolyser having no diaphragm was compared. The biological activity of solutions containing molecular chlorine, hypochlorous acid, and hypochlorite ions was determined by their effect on the growth of E. coli cells. After a 5-min incubation of cells with each of the oxidizers, the bacterial growth stopped and was not restored during one day. The conclusion is made that the oxidizers irreversibly disturb the barrier properties of cell membranes and, in some cases, destroy cells. In model solutions, as well as in solutions treated after heating on a water bath or after the addition of sodium thiosulfate, a delay in the start of E. coli growth occurs. After the lag-phase, the repair of cells sets on, and after a day the optical density of cells increases and approaches the control.     

Intragastric generation of antimicrobial nitrogen oxides from saliva--physiological and therapeutic considerations

Salivary nitrite is suggested to enhance the antimicrobial properties of gastric juice by conversion to nitric oxide (NO) and other reactive nitrogen intermediates in the stomach. Intubated patients exhibit extremely low gastric levels of NO, because they do not swallow their saliva. The present investigation was designed to examine the antibacterial effects of human saliva and gastric juice. Furthermore, we studied a new mode of NO delivery, involving formation from acidified nitrite, which could prevent bacterial growth in the gastric juice of intubated patients in intensive care units. The growth of Escherichia coli ATCC 25922 and the formation of NO and nitroso/nitrosyl species were determined after incubation of gastric juice with saliva from healthy volunteers that was rich (nitrate ingestion) or poor (overnight fasting) in nitrite. In a stomach model containing gastric juice from intubated patients, we inserted a catheter with a silicone retention cuff filled with ascorbic acid and nitrite and determined the resulting antibacterial effects on E. coli and Candida albicans. Saliva enhanced the bactericidal effect of gastric juice, especially saliva rich in nitrite. Formation of NO and nitroso/nitrosyl species by nitrite-rich saliva was 10-fold greater than that by saliva poor in nitrite. In our stomach model, E. coli and C. albicans were killed after exposure to ascorbic acid and nitrite. In conclusion, saliva rich in nitrite enhances the bactericidal effects of gastric juice, possibly through the generation of reactive nitrogen intermediates, including NO. Acidified nitrite inside a gas-permeable retention cuff may be useful for restoring gastric NO levels and host defense in critically ill patients.     

Isolation of sodium chloride tolerant cell lines and plants in finger millet

Sodium chloride tolerant cell lines of finger millet were isolated from embryogenic cultures growing on MS medium supplemented with picloram (2 mg I^-1), kinetin (0.1 mg l^-1) and sodium chloride (1 %) at the end of 6 passages. The sodium chloride tolerant cell lines showed better growth in comparison with control at all concentrations of sodium chloride tested, with optimum growth at 0.25 % NaCl. When the tolerant lines were grown for 3 passages in absence of NaCl, the growth was lower than that of the tolerant lines tested immediately at the end of 6 passages of selection. NaCl tolerant calli had more Na¹ in comparison with control and they regenerated plants in presence of 1 % NaCl, while the control lines failed to differentiate. When screened in a hydroponics system with 1 % NaCl, the tolerant plants grew to maturity while the control plants failed to grow.     

Use of Gradient Plates To Study Combined Effects of Temperature, pH, and NaCl Concentration on Growth of Monascus ruber van Tieghem, an Ascomycetes Fungus Isolated from Green Table Olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35°C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30°C but not at 35°C. Growth was better at 30 and 25°C as judged by the larger extent of the plates covered by mycelium compared with that at 35°C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35°C, whereas at 35°C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

The effect of incubation temperature, sodium chloride and ascorbic acid on the growth kinetics of Aeromonas hydrophila

The effects of temperature, sodium chloride and ascorbic acid on the aerobic growth kinetics of a clinical strain of Aeromonas hydrophila were evaluated. At 5°C, ascorbic acid (1 mmol l^-1) and sodium chloride (3% w/v) inhibited the growth of the organism. At 10°C, ascorbic acid depressed only the maximum population densities (A) by approximately 2 log cycles, but not maximum specific growth rate (μ_m) or the lag time (Λ). On the contrary, NaCl caused A to increase, with the effect being greatest when the NaCl content was 1.5%. Temperature increase from 10 to 15°C resulted in an approximate doubling of μ_m and unexpectedly an apparent increase in Λ However, this apparent increase resulted from the particular manner in which the lag phase was mathematically calculated.     

Combined treatment of preimplantation mouse embryos in vitro with sodium nitrite and X-rays

Summary Man takes up nitrite in a considerable amount. Effects of nitrite on DNA have been reported; therefore, interaction between nitrite and radiation might be possible.Preimplantation mouse embryos in vitro were treated with a combination of sodium nitrite (1 mM or 2.5 mM) and X-rays (0.94 Gy) in order to obtain some information whether radiation risk is influenced by the presence of nitrite. The microscopic visible development up to 144 h post conceptionem (h p.c.), the number of cell nuclei, and the number of micronuclei were determined.None of the experimental results gives any indication that radiation risk is influenced by nitrite. All effects after combined treatment correspond to the sum of the single effects.