Inactivation of bacteria by Purogene

The bacteriocidal efficacy of Purogene, a stabilized aqueous solution of chlorine dioxide (ClO2) was examined using bacteria of concern to public health. The organisms tested were: Escherichia coli, Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella pneumoniae, Streptococcus pyogenes Group A, Salmonella typhimurium and Bacillus subtilis. The test organisms responded differently to inactivation by Purogene. At least a 4 log reduction in bacterial counts was noted when Purogene was applied at a concentration of 0.75 mg/l. Since Purogene is a stabilized complex, it was necessary to provide a chemical environment suitable for the release of ClO2 in this solution. This was done by varying the pH of Purogene from 3.5 to 8.6 (pH of Purogene is 8.6) while keeping the pH of the experimental medium constant (pH 7.0). The results showed that Purogene was most efficacious at the lowest pH tested (pH 3.5). This indicates that as chlorine dioxide solutions were reduced to chlorite (which predominates at pH 8.6), their bacteriocidal efficacy was reduced, suggesting free chlorine dioxide as the active disinfecting species.     

Plasma membrane damage to Candida albicans caused by chlorine dioxide (ClO2)

Aim:  To investigate the plasma membrane damage of chlorine dioxide (ClO₂) to Candida albicans ATCC10231 at or below the minimal fungicidal concentration (MFC).
Methods and Results:  ClO₂ at MFC or below was adopted to treat the cell suspensions of C. albicans ATCC10231. Using transmission electron microscopy, no visible physiological alteration of cell shape and plasma membrane occurred. Potassium (K⁺) leakages were significant; likewise, it showed time- and dose-dependent increases. However, adenosine triphosphate (ATP) leakages were very slight. Research shows that when 99% of the cells were inactivated, the leakage was measured at 0·04% of total ATP. Compared with the mortality-specific fluorescent dye of DiBAC₄(3), majority of the inactivated cells were poorly stained by propidium iodide, another mortality-specific fluorescent dye which can be traced by flow cytometry.
Conclusion:  At or below MFC, ClO₂ damages the plasma membranes of C. albicans mainly by permeabilization, rather than by the disruption of their integrity. K⁺ leakage and the concomitant depolarization of the cell membrane are some of the critical events.
Significance and Impact of the Study:  These insights into membrane damages are helpful in understanding the action mode of ClO₂.     

Bactericidal and sporicidal performance of a polymer-encapsulated chlorine dioxide-coated surface

Aims:  To investigate the physical characteristics and the bactericidal and sporicidal potential of a polymer-encapsulated ClO₂ coating.
Methods and Results:  An antimicrobial coating based on polymer-encapsulated ClO₂ was developed. A low viscosity, water/oil/water double emulsion coating was formulated for easy on-site application. Escherichia coli , Pseudomonas aeruginosa , Bacillus subtilis and Staphylococcus aureus were applied onto the coating to study the bactericidal capabilities of the coating. The bactericidal performance of the coating increased when the contact time with the tested bacteria increased. Over 99% of the E. coli , Ps. aeruginosa , B. subtilis were killed with a contact time of 30 min. Although endospores of B. subtilis are more resistant, about 75% of the spores were killed after 72 h on the coating. Moreover, a sustained release of gaseous ClO₂ was achieved to maintain about 90% removal of B. subtilis with a 10-min contact time during a 28-day study period. The coating also exhibits antiadhesive properties against bacteria.
Conclusions:  A polymer-encapsulated ClO₂ coating with sustained release of ClO₂ and promising bactericidal and sporicidal features was tested for 28 days.
Significance and Impact of the Study:  This study provides a new direction for developing polymer-encapsulated ClO₂ coatings that possess persistent bactericidal and sporicidal properties.     

Use of superabsorbent polymer gels for surface decontamination of Bacillus anthracis spores

Aims:  This study evaluated the inactivation of Bacillus anthracis Vollum spores dried on a nonporous surface using a superabsorbent polymer (SAP) gel containing commercially available liquid decontaminants.
Methods and Results:  The first phase determining the availability of the liquid decontaminant within the SAP showed that the SAP gel containing pH-adjusted sodium hypochlorite (NaOCl) inhibited B. anthracis growth while the water control SAP gel had no affect on growth. For testing surface decontamination, B. anthracis spores were dried onto steel coupons painted with chemical agent resistant coating and exposed to SAP containing either pH-adjusted NaOCl, chlorine dioxide (ClO₂) or hydrogen peroxide/peracetic acid (H₂O₂/PA) for 5 and 30 min. At contact times of both 5 and 30 min, all of the SAP gels containing pH-adjusted NaOCl, ClO₂ or H₂O₂/PA inactivated B. anthracis spores at levels ranging from 2·2 to ≥7·6 log reductions.
Conclusions:  Incorporation of three commercially available decontaminant technologies into a SAP gel promotes inactivation of B. anthracis spores without observable physical damage to the test surface.
Significance and Impact of the Study:  This work provides preliminary data for the feasibility of using SAP in inactivating B. anthracis spores on a nonporous surface, supporting the potential use of SAP in surface decontamination.     

Chlorate-resistant rose cells: influx, efflux and reduction of [36Cl]-chlorate

Clones of Rosa damascena Mill. cv. Gloire de Guillan, selected for growth in solid medium containing 56 m M NaClO₃, were studied to determine the reason for their resistance to this toxic salt. The cells grew on medium containing nitrate as the only nitrogen source, and they synthesized nitrate reductase (EC 1.6.6.2) in the presence of nitrate. The cells were resistant in the presence of nitrate. However, their resistance was greatly increased by the presence of glutamate in the medium. The cells took up [³⁶Cl]-ClO₃- and reduced it to ClO₂⁻, but the fraction of ClO₃⁻ that they reduced under our experimental conditions was less than that reduced by wild type. The slower production of ClO₂⁻ apparently accounted for the resistance of the cells to ClO₃⁻. We suggest several possible reasons for the low rate of reduction of ClO₃⁻.     

A precise potentiometric method for determination of algal activity in an open CO2 system

Abstract. The activity of the green alga Scenedesmus obliquus was studied in simplified nutrient solutions (20 mol m₋₃ NaNO₃, 20 mol m₋₃ NH₄C1, 20 mol m₋₃ NH₄NO₃, and 20 mol m₋₃ NaCl, respectively) at 25 °C. The experiments were performed under welldefined incident photon density fluxes ranging from 10 to 200 μmol m₂ s₋₁, Light-dependent changes in pH and alkalinity (A) were followed by means of a potentiometric method using a glass electrode. In the experiments, carbon dioxide with known partial pressure was bubbled through the algal suspension, and during dark periods ul intervals of 1 h, the solution was allowed to equilibrate with the gas phase. This technique was applied to calculate equilibrium values of pH and alkalinity at regular intervals during a 12-h period. Results obtained in NaNO₃, solution show a linear increase in A with time, at each level of illumination studied. After an initial drop, A also increases in NH₄NO₃, solution in a similar way to that in NaNO₃ solution. The change in A with time was also found to increase linearly with the photon density flux studied and no saturation level could be defined. In experiments in NaCl solution, no changes in A were registered while measurements in NH₄Cl solution showed a decrease in A with time.     

The microbial flora of herring fillets after storage in carbon dioxide, nitrogen or air at 2°C

Herring fillets from the Baltic Sea were stored in glass vessels in air, nitrogen or carbon dioxide (CO₂) at 2°C and the microbial development was studied. The microbiological shelf-life of the herring (the time to reach 10⁷ organisms/g) was prolonged by a factor of 3.5 in CO₂ as compared to air. The corresponding factor in nitrogen was 1.5. The microflora of fresh and spoiled herring was classified. The initial microflora was dominated by coryneforms, Flavobacterium spp., Moraxella -like organisms and Pseudomonas spp. The spoilage-flora in air (after 9 d) was dominated by Pseudomonas spp. and Moraxella -like organisms, and in nitrogen (14 d) Enterobacteriaceae, Vibrionaceae and Lactobacillus spp. were dominant. Homofermentative Lactobacillus spp. were the only organisms isolated from fillets stored in CO₂ (28 d). It was concluded that storing fresh fish in pure CO₂ at low refrigeration temperatures is a method with industrial potential. The method (1) improves the microbiological stability and (2) reduces the microbiological health hazards of the fish.     

Ecophysiological characterization of common food-borne fungi in relation to pH and water activity under various atmospheric compositions

The combined effects of pH, water activity (a_w), oxygen (O₂) and carbon dioxide (CO₂) levels on growth and sporulation of 10common food-borne fungi were studied. The use of a multivariate statistical method (PLS) for the analysis of data showed that the fungi could be grouped according to their physiological response to changes in the four tested factors. Carbon dioxide, a_w and pH were found to be the most significant factors describing differences and similarities among the fungi. Maximal inhibitory effect of elevated levels of CO₂ (5–25%) and decreased a_w (0·99–0·95) varied among the 10 species from 6 to 77% and from 52 to 100%, respectively. Sporulation of the fungi was sensitive to all tested factors. Furthermore, interaction of CO₂ and a_w displayed a significant effect on sporulation. It was shown that different fungal species associated with the same ecosystem responded similarly to changes in the tested factors. Thus, fungi which are not phylogenetically related may be physiologically related or show a common strategy of life.     

Efficacy of certain disinfectants against infectious pancreatic necrosis virus

The virucidal properties of iodophor, chlorine (sodium hypochlorite), formalin, thimerosal (organic mercurial compound), malachite green, and acriflavine were tested on infectious pancreatic necrosis virus (IPNV). Iodine and chlorine showed good activity, but efficacy depended on the concentration of virus, the presence of organic matter (calf serum), and water _pH. Water hardness (0-300 mg 1⁻¹ as CaCO₃) did not affect virucidal activity. In a 5 min exposure, 4 mg 1⁻¹ available iodine inactivated 10^3.9 TCID₅₀ m1⁻¹ IPNV but 16 mg 1⁻¹ iodine were needed for inactivation of 10^6.3TCID₅₀m1⁻¹. The addition of 0-5% calf serum significantly reduced the iodine concentration and the virucidal activity. In comparison, 4 mg 1⁻¹ chlorine were needed to inactivate 10⁴⁶ TCID₅₀ m1⁻¹ IPNV in 5 min. However, the addition of 0-07 % serum greatly reduced the chlorine concentration and extended the virucidal contact time to 30 min or more. IPNV at 10^6.3 TCID₆₀ m1⁻¹ was not inactivated by exposures for 60 min to 0-2% formalin, 10 min to 0-2% thimerosal, 60 min to 5 mg 1⁻¹ malachite green, or 20 min to 500 mg 1⁻¹ acriflavine. However, acriflavine at 0-5 mg 1⁻¹ in cell culture media prevented the development of cytopathology caused by IPNV and may be useful in the treatment of the disease.     

Comparison of quantitative and qualitative methods of detecting hydrogen peroxide produced by human vaginal strains of lactobacilli

A quantitative method was developed for the measurement of micromolar quantities of H₂O₂ produced in Rogosa broth and peptonized milk broth by vaginal strains of lactobacilli isolated from women. The production of substantial amounts reproducibly was dependent on the growth of the organisms in acid media (pH ≤6.0) under anaerobic or micro-aerophilic conditions with continuous agitation. The addition to the media of the enzyme inhibitor, 3-amino-l,2,4-triazole, with or without catalase sometimes induced the production of H₂O₂ especially in non-agitated cultures. However, other agents such as concanavalin and o -dianisidine had no enhancing effect, and catalase or peroxidase alone completely inhibited H₂O₂ production.
The H₂O₂ produced in the acid media was stable for more than a month at 5°C but not in media at pH ≥ 7.0. Of five strains of lactobacilli tested by the quantitative method and by a chromogenic qualitative method (Rogosa-catalase or -peroxidase agar), three consistently produced H₂O₂ measurable by the former method, but none did so after growth of the organisms on Rogosa-catalase/peroxidase agar which suggested that the qualitative method was unreliable. The fact that H₂O₂ was produced in substantial quantities by some strains and not at all by others enabled H₂O₂-producers and non-producers to be distinguished easily.     

The Measurement of Spinal Cord Tissue pH Using a Diffusible, Lipid-Soluble, pH-Sensitive Fluorescent Indicator

Abstract: Spinal cord tissue pH was measured in cats at normocapnia, hypocapnia, hypercapnia and death from anoxia using a pH-sensitive fluorescent indicator (umbelliferone) with both molecular and ionic fluorophors. A ratio analysis of the indicator's calibrated 450 nm fluorescent tissue clearance curves from 340 and 370 nm excitation permitted direct in vivo tissue pH determinations. Fifteen animals were divided into three equal groups according to different arterial carbon dioxide tensions (P_a co₂):five animals at P_a co₂ 20, five animals P_a co₂ 40 and five animals P_a co₂ 60 torr. Spinal cord tissue pH varied linearly with arterial pH, but within narrower limits. These values (arterial versus cord pH) were: 7.46 versus 7.15; 7.21, 7.09; and 7.04, 7.00. At death from hypoxemia the arterial pH fell to 6.99 and spinal cord to 6.67. The clearance curves of umbelliferone in spinal cord varied according to P_a co₂ and appeared to reflect spinal cord blood flow.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.     

Relationship of environmental pCO2 to selected blood parameters of Oncorhynchus kisutch (Walbaum)

Blood samples from sub-adult coho salmon ( Oncorhynchus kisutch ) were correlated with environmental conditions. Carbon dioxide tensions, pH and bicarbonate levels were determined in the blood for fish acclimated to environmental pCO₂ levels of 2.49 and 2.74 mmHg. Fish were removed from the high pCO₂ environment and placed in one with a low pCO₂. Their blood pH and pCO₂ were monitored during the course of acclimation. After 27 days of acclimation to a low pCO₂ environment, a group of fish was sampled and compared to a group of fish acclimated to a high pCO₂ environment. It was found that pCO₂ in the environment affects the rate of diffusion of carbon dioxide from the blood to the water. Under the environmental conditions prescribed in these experiments no significant change in bicarbonate was noticed. It is postulated that a bicarbonate chloride exchange is not a major pathway for the removal of carbon dioxide from the blood of O. kisutch .     

The Effect of Temperature, Pressure and Chlorine Concentration of Spray Washing Water on Numbers of Bacteria on Lamb Carcases

Combined analysis of three experiments showed that when lamb carcases with initial bacterial numbers of between logi₁₀3.29 and 4.22/cm² were spray washed, statistically significant reductions in bacterial numbers of log₁₀O.5 were obtained when the spray wash water temperature was > 57°C, and reductions of log₁₀1.0 were obtained when the temperature was ≥ 80°C. Reductions at all temperatures were enhanced by log₁₀0.66 when the water contained 30 µg/ml chlorine, but increasing the concentration to 450 µg/ml reduced bacterial numbers only by a further log₁₀0–29. At highly contaminated sites increasing the duration of spraying from 30 to 120 s significantly increased the reductions obtained when water containing added chlorine was used. Reductions in bacterial numbers after spray washing with pressures of 3.5, 5.6. 7.7 kg/cm² were not significantly different.     

Determination of dissolved carbon dioxide by coulometric titration in modified atmosphere systems

The solubility of carbon dioxide (CO₂) in microbiological media at different pH values, water activities ( a_w ), temperatures, buffering capacities and ratios of headspace to media volumes was determined by using a coulometer. Buffering capacity and ratio of headspace to media volume were shown to be the major factors influencing the solubility of CO₂ in modified atmosphere model systems. The growth inhibitory effects of different dissolved CO₂ concentrations (0–50 μmol ml^-1) were determined for Pseudomonas fragi at 8°C and 22 C. Pseudomonas fragi was shown to be strongly affected by the CO₂ concentration in the media. A carbon dioxide concentration of 40 μmol ml^-1 was needed to inhibit Ps. fragi at 8°C. The importance of measuring dissolved CO₂ concentrations in modified atmosphere packaging applications was shown and the coulometer proved to be an excellent tool for this purpose.     

Vitamin B12 Production by the Gastro-intestinal Microflora of Baboons Fed either a Vitamin B12 Deficient Diet or a Diet Supplemented with Vitamin B12

The vitamin B₁₂-producing capacity of micro-organisms isolated from baboon faeces and gastric contents was measured using Lactobacillus leichmannii . The animals were fed either a diet deficient in or supplemented with vitamin B₁₂ (controls). Samples of gastric and small intestinal contents, obtained at laparotomy from two young vitamin B₁₂-deprived baboons, contained varying quantities of vitamin B₁₂. Many of the organisms isolated from these aspirates produced vitamin B₁₂ in vitro . The highest levels of vitamin B₁₂ were produced by anaerobic organisms. Gastric juice samples from vitamin B₁₂ deprived and control baboons contained similar types of organisms with like vitamin B₁₂-producing capacity. The vitamin B₁₂ content, pH and total bacterial counts of gastric juice samples aspirated after a 6 h fast from vitamin B₁₂ deprived baboons were not significantly different from those of the control animals. The pH values of gastric juice samples aspirated 18 h after feeding, however, were significantly lower than those of 6 h fasting samples in both groups. The mean vitamin B₁₂ levels in the total volumes of gastric juice aspirated after each fasting period were similar. The possible involvement of the gastrointestinal flora in the vitamin B₁₂ status of the baboon is discussed.     

Nitrite in the root zone and its effects on ion uptake and growth of wheat seedlings

The immediate and posteffects of various concentrations of NaNO₂ on ion uptake of wheat ( Triticum aestivum L. cv. GK Öthalom) seedlings were studied at different pH values. Without pretreatment, the higher the concentration of NaNO₂ the greater was the decrease in uptake of K⁺ into the roots, both at pH 4 and pH 6. At pH 6 but not at pH 4 the reverse was true when the seedlings were pretreated with NaNO₂. Due to the high Na⁺ content of the roots, an effect of Na⁺ in this process cannot be excluded. Nitrite was taken up by the roots more rapidly than nitrate. Nitrite at 0.1 m M in the medium induced the development of an uptake system for both NO⁻₂ and NO⁻₃ in wheat roots. At higher concentrations pretreatment with NO⁻₂ decreased NO⁻₃ uptake by the roots, but NO₃ did not inhibit the uptake of NO₂. The toxic effect of NO⁻₂ was strongly pH dependent. Lower pH of the external solution led to an increased inhibition by NO⁻₂ of both ion uptake and growth of seedlings. The inhibitory effect of NO⁻₂ differed considerably for roots and shoots. The roots and especially the root hairs were particularly sensitive to NO⁻₂ treatment.     

Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP

Escherichia coli shifted from broth at external pH (pH₀) 7·0 to pH₀ 7·0 broth plus glucose rapidly induced marked acid tolerance which also appeared, albeit to a lesser extent, plus maltose, sucrose or lactose. Tolerance appeared without the medium pH becoming acidic. Tolerance was most substantial when glucose was added at pH₀ 7·0 but was also appreciable at pH₀ 7·5, 8·0 and 8·5. Induction of tolerance by glucose was markedly reduced by cyclic AMP and essentially abolished plus NaCl or sucrose ; the induction process was also reduced but not fully inhibited by chloramphenicol, tetracycline and nalidixic acid. Glucose-induced organisms showed less acid damage to DNA and β-galactosidase and it is likely that this is because glucose induces a new pH homeostatic mechanism which keeps internal pH close to neutrality at acidic pH₀. In conclusion, it is clear that glucose induces a novel acid tolerance response in log-phase E. coli at pH₀ 7·0 ; it is now known that induction of this response involves the functioning of extracellular induction components including an extracellular induction protein.     

Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3-chlorobenzoate

Abstract A defined 3-chlorobenzoate-degrading methanogenic consortium was constructed by recombining key organisms isolated from a 3-chlorobenzoate-degrading methanogenic sludge enrichment. The organisms comprise a three-tiered food chain which includes: (1) reductive dechlorination of 3-chlorobenzoate; (2) oxidation of benzoate to acetate, H₂ and CO₂; (3) removal of H₂ plus CO₂ by conversion into methane. The defined consortium, consisting of a dechlorinating organism (DCB-1), a benzoate degrader (BZ-1) and a lithotrophic methanogen ( Methanospirillum strain PM-1) grew well in a basal salts medium supplemented with 3-chlorobenzoate (3.2 mM) as the sole energy source. The chlorine released from the aromatic ringe was recovered in stoichiometric amounts as the chloride ion. The reducing power required for reductive dechlorination was obtained from the hydrogen produced in the acetogenic oxidation of benzoate. One-third of the benzoate-derived hydrogen was recycled via the reductive dechlorination of 3-chlorobenzoate, indicating that the consortium operated as a food web rather than a food chain.     

Low external pH prevents cell elongation but not multiplication of embryogenic carrot cells

Embryogenic cultures of cultivated carrot ( Daucus crota cv. Scarlet Nantes) were initiated from seedling hypocotyls on hormone-containing nutrient medium and from wounded zygotic embryos on hormone-free medium. Both of these cultures were maintained with continuous multiplication as unorganized, embryogenic cell masses on hormone-free medium at pH 4.0, containing NH⁺₄ as the sole nitrogen source. When grown on hormone-free medium at pH 4.0, neither culture contained any elongated cells. Virtually all cells were densely cytoplasmic and nearly spherical. Some cells were enlarged, not densely cytoplasmic, but always spherical. When either culture was transferred to an auxin-containing medium at pH 5.8, numerous elongated cells were produced. Elongated cells were observed when either naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was used, and whether the nitrogen source was NH⁺₄ alone or a combination of NH⁺₄ and NO⁻₃. Elongated cells were more abundant when a combined nitrogen source was used. When cultures containing elongated cells were transferred to and multiplied on hormone-free or hormone-containing medium buffered at pH 4.0, all elongated cells disappeared after 2 weeks. No elongated cells were observed in any of the lines tested at pH 4.0. These results clearly show that it was the pH of the culture medium and not the presence or absence of an auxin or the nitrogen source(s) that permitted or prevented cell elongation in the embryogenic cultures tested.