Ozonized Water in Microbial Control: Analysis of the Stability, In Vitro Biocidal Potential, and Cytotoxicity

O3 dissolved in water (or ozonized water) has been considered a potent antimicrobial agent, and this study aimed to test this through microbiological and in vitro assays. The stability of O3 was accessed following modifications of the physicochemical parameters of water, such as the temperature and pH, with or without buffering. Three concentrations of O3 (0.4, 0.6, and 0.8 ppm) dissolved in water were tested against different microorganisms, and an analysis of the cytotoxic effects was also conducted using the human ear fibroblast cell line (Hfib). Under the physicochemical conditions of 4 °C and pH 5, O3 remained the most stable and concentrated compared to pH 7 and water at 25 °C. Exposure to ozonized water resulted in high mortality rates for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans. Scanning electron micrograph images indicate that the effects on osmotic stability due to cell wall lysis might be one of the killing mechanisms of ozonized water. The biocidal agent was biocompatible and presented no cytotoxic effect against Hfib cells. Therefore, due to its cytocompatibility and biocidal action, ozonized water can be considered a viable alternative for microbial control, being possible, for example, its use in disinfection processes.

Cryopreservation of Canine Erythrocytes Using Dimethyl Sulfoxide, Polyethylene Glycol and Sucrose

Cryoprotective properties of combined media of permeable (dimethyl sulfoxide) and impermeable (polyethylene glycol with m. w. 1500) cryoprotective agents during rapid cooling in liquid nitrogen of canine erythrocytes using saline and sucrose-saline media have been investigated. It was found that the use of combined solutions of cryoprotective agents based on polyethylene glycol with m.w. 1500 (15%) and dimethyl sulfoxide (2.5–10%) in saline was not quite effective for cryopreservation of canine erythrocytes. Reducing the salt concentration and adding cell-impermeable sucrose to the cryopreservation medium increase the preservation of erythrocytes after warming. The best cryoprotective properties for canine erythrocytes were demonstrated by 10% dimethyl sulfoxide based on sucrose-saline medium, with high preservation of cells after freeze-warming, mechanical and osmotic stability of warmed erythrocytes. This indicates the possibility of a long-term storage and use of cryopreserved canine erythrocytes for transfusions.

Plasmodium vivax infection compromises reticulocyte stability

The structural integrity of the host red blood cell (RBC) is crucial for propagation of Plasmodium spp. during the disease-causing blood stage of malaria infection. To assess the stability of Plasmodium vivax-infected reticulocytes, we developed a flow cytometry-based assay to measure osmotic stability within characteristically heterogeneous reticulocyte and P. vivax-infected samples. We find that erythroid osmotic stability decreases during erythropoiesis and reticulocyte maturation. Of enucleated RBCs, young reticulocytes which are preferentially infected by P. vivax, are the most osmotically stable. P. vivax infection however decreases reticulocyte stability to levels close to those of RBC disorders that cause hemolytic anemia, and to a significantly greater degree than P. falciparum destabilizes normocytes. Finally, we find that P. vivax new permeability pathways contribute to the decreased osmotic stability of infected-reticulocytes. These results reveal a vulnerability of P. vivax-infected reticulocytes that could be manipulated to allow in vitro culture and develop novel therapeutics.

About the Mechanism of Mammalian Erythrocytes Osmotic Stability

The peculiarities of the effect of hypertonic shock and hypotonic stress on erythrocytes of different species of mammals (human, bull, horse, rabbit, dog, rat) have been investigated. Based on the results of correlation analysis (using the Spearman’s rank correlation coefficient), the relationship between osmotic sensitivity of mammalian erythrocytes and the well-known structural and functional characteristics of these cells was assessed. The paper presents and analyzes the significant relationships. Under hypotonic stress of mammalian erythrocytes, the values of the threshold concentration of NaCl and the one of osmotic fragility were found to correlate with the size of cells (diameter). Under hypertonic shock of mammalian erythrocytes, the values of the threshold concentrations of NaCl and that of hemolysis of cells in a medium containing 4.0 mol/L NaCl correlated with the membrane permeability to water. Mammalian erythrocytes with a high value of the coefficient of diffusion water transport due to the protein channels are more resistant to hypertensive shock.

Plasmodium vivax infection compromises reticulocyte stability.

The structural integrity of the host red blood cell (RBC) must be maintained for propagation of Plasmodium spp. during the disease causing blood-stage of malaria infection. Plasmodium vivax infection is restricted to reticulocytes. To assess the impact of P. vivax infection on reticulocyte stability, we developed a flow cytometry-based assay capable of measuring osmotic stability within heterogeneous RBC populations. We found that P. vivax preferred young reticulocytes are more osmotically stable than older reticulocytes and normocytes, and P. vivax infection decreased reticulocyte stability to levels observed for RBC disorders that cause hemolytic anemia. Moreover, P. vivax reticulocyte destabilization was more significant than P. falciparum normocyte destabilization. Finally, we found that P. vivax new permeability pathways contribute to the decreased osmotic stability of infected-reticulocytes. These results reveal a key vulnerability of P. vivax that could be manipulated to yield both in vitro culture and novel therapeutics.