Traditional Plate Count Research Articles

The Complex Essential Oils Highly Control the Toxigenic Fungal Microbiome and Major Mycotoxins During Storage of Maize.

The contamination of maize with fungi and subsequent mycotoxins is a pivotal and long-standing safety concern in the maize industry. In this study, the inhibitory effects of the complex essential oils (cinnamaldehyde, citral, eugenol, and menthol, 3:3:2:2, v/v) on fungal growth and mycotoxins production in stored maize were evaluated using traditional plate counting, internal transcribed spacer 2 (ITS2) sequencing and liquid chromatography–tandem mass spectrometry. Complex essential oils (0.02%) significantly (p < 0.05) reduced the total fungi counts and the content of aflatoxin B1, zearalenone, and deoxynivalenol in stored maize during 12 months of storage, and were more effective than propionic acid (0.2%). The fungal diversity of the control group was the highest with 113 operational taxonomic units. During storage of maize kernels, Aspergillus, Fusarium, Wallemia, Sarocladium, and Penicillium were main genera. At 0–6 months, the fungal diversity was high and Fusarium was predominant genus. However, at 7–11 months, the fungal diversity was low and Aspergillus was predominant genus. During the later stages of storage, the prevalence of Aspergillus in maize treated with essential oils was significantly lower than (p < 0.05) that observed in the propionic acid treated and control samples. The results of this study suggest that the complex essential oils may be employed successfully to control toxigenic fungi and subsequent contamination with mycotoxins in maize.

Flow cytometry, a powerful novel tool to rapidly assess bacterial viability in metal working fluids: Proof-of-principle.

Metalworking fluids (MWF) are water- or oil-based liquids to cool and lubricate tools, work pieces and machines, inhibit corrosion and remove swarf. One of the major problems in the MWF industry is bacterial growth as bacterial enzymes can cause MWF degradation. In addition, bacteria can form biofilms which hamper the functioning of machines. Last but not least, some bacterial by-products are toxic (e.g. endotoxins) and present potential health risks for metalworking machine operators via the formation of aerosols. Therefore, a novel fast yet accurate analytical method to evaluate and predict the antibacterial capacity of MWF would be an important asset. As such a tool is currently lacking, the present study aimed to develop a protocol based on flow cytometry (FCM) to assess the antibacterial potential of newly developed MWF independent of bacterial growth. Results of this novel method were compared to a biochallenge test currently used in MWF industry and also to traditional plate counts. Our results represent a proof-of-principle that FCM can reliably predict the antibacterial capacity of MWF already within one day of incubation with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis, being substantially faster than the current growth-based methods.

Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM® and Bifidobacterium animalis subsp. lactis Bl-04 ® via Chip-Based Digital PCR.

The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR) to enumerate Bifidobacterium animalis subsp. lactis Bl-04 and Lactobacillus acidophilus NCFM both as single strains and in combination. Primers and probes were designed to differentiate the target strains against other strains of the same species using known single copy, genetic differences. The assay was optimized to include propidium monoazide pre-treatment to prevent amplification of DNA associated with dead probiotic cells as well as liberation of DNA from cells with intact membranes using bead beating. The resulting assay was able to successfully enumerate each strain whether alone or in multiplex. The cdPCR method had a 4 and 5% relative standard deviation (RSD) for Bl-04 and NCFM, respectively, making it more precise than plate counts with an industry accepted RSD of 15%. cdPCR has the potential to replace traditional plate counts because of its precision, strain specificity and the ability to obtain results in a matter of hours.

The Contamination Degree of Characteristic Fungi in Jet Fuel Detected by ATP Bioluminescence

Adenosine Triphosphate (ATP) lysate of the characteristic fungi in jet fuel was screened and three fluorescein-luciferase systems were compared in the paper. The correlation between ATP bioluminescence and traditional plate count for measuring the number of microorganism was also investigated and then a method of detecting the contamination degree of the characteristic fungi of jet fuel by ATP bioluminescence were initially established. The results show that the effect of ATP extraction taking surfactant Benzyldimethylhexadecylammonium chloride (BAC) as the microbial lysate was optimal and the optimal concentration and the action time were 0.15% and 30s respectively; The fluorescein-luciferase system after screening has a good detection limit, up to 10-15mol ATP; using ATP bioluminescence and traditional plate method to measure the microbial quantity, they have good correlation. Utilizing ATP bioluminescence to detect the contamination degree of the characteristic fungi in jet fuel can shorten the test time to 10min, which is suitable for rapid detection and has good application prospects.

Effectiveness of UV-C light assisted by mild heat on Saccharomyces cerevisiae KE 162 inactivation in carrot-orange juice blend studied by flow cytometry and transmission electron microscopy

The aim of this study was to analyze the effectiveness of UV-C light (0–10.6 kJ/m2) assisted by mild heat treatment (50 °C) on the inactivation of Saccharomyces cerevisiae KE 162 in peptone water and fresh carrot-orange juice blend (pH: 3.8; 9.8°Brix; 707 NTU; absorption coefficient: 0.17 cm−1). Yeast induced damage by single UV-C and mild heat (H) and the combined treatment UV-C/H, was investigated by flow cytometry (FC) and transmission electron microscopy (TEM). When studying induced damage by FC, cells were labeled with fluorescein diacetate (FDA) and propidium iodide (PI) to monitor membrane integrity and esterase activity. UV-C/H provoked up to 4.7 log-reductions of S. cerevisiae; whereas, only 2.6–3.3 log-reductions were achieved by single UV-C and H treatments. FC revealed a shift with treatment time from cells with esterase activity and intact membrane to cells with permeabilized membrane. This shift was more noticeable in peptone water and UV-C/H treated juice. In the UV-C treated juice, double stained cells were detected, suggesting the possibility of being sub-lethally damaged, with compromised membrane but still metabolically active. TEM images of treated cells revealed severe damage, encompassing coagulated inner content, disorganized lumen and cell debris. FC and TEM provided additional information regarding degree and type of damage, complementing information revealed by the traditional plate count technique.

Survival of Spoilage and Pathogenic Microorganisms on Cardboard and Plastic Packaging Materials.

The aim of this work was to study the interaction of corrugated and plastic materials with pathogenic and spoiling microorganisms frequently associated to fresh produce. The effect of the two packaging materials on the survival during the storage of microorganisms belonging to the species Escherichia coli, Listeria monocytogenes, Salmonella enteritidis, Saccharomyces cerevisiae, Lactobacillus plantarum, Pseudomonas fluorescens, and Aspergillus flavus was studied through traditional plate counting and scanning electron microscopy (SEM). The results obtained showed that cardboard materials, if correctly stored, reduced the potential of packaging to cross-contaminate food due to a faster viability loss by spoilage and pathogenic microorganisms compared to the plastic ones. In fact, the cell loads of the pathogenic species considered decreased over time independently on the inoculation level and packaging material used. However, the superficial viability losses were significantly faster in cardboard compared to plastic materials. The same behavior was observed for the spoilage microorganisms considered. The SEM microphotographs indicate that the reduction of superficial contamination on cardboard surfaces was due to the entrapping of the microbial cells within the fibers and the pores of this material. In addition, SEM data showed that the entrapped cells were subjected to more or less rapid lyses, depending on the species, due to the absence of water and nutrients, with the exception of molds. The latter spoilers were able to proliferate inside the cardboard fibers only when the absorption of water was not prevented during the storage. In conclusion, the findings of this work showed the reduction of cross-contamination potential of corrugated compared to plastic packaging materials used in fruit and vegetable sector. However, the findings outlined the importance of hygiene and low humidity during cardboard storage to prevent the mold growth on packaging.

Emerging Biorecognition and Transduction Schemes for Rapid Detection of Pathogenic Bacteria in Food.

The presence of unsafe levels of microorganisms in food constitutes a growing economic and public health problem that necessitates new technology for their rapid detection along the food continuum from production to consumption. While traditional techniques are reliable, there is a need for more sensitive, selective, rapid, and cost-effective approaches for food safety evaluation. Methods such as microbiological counts are sufficiently accurate and inexpensive, and are capable of determining presence and viability for most pathogens. However, these techniques are time consuming, involve destructive sampling, and require trained personnel and biosafety-certified facilities for analysis. Molecular techniques such as the polymerase chain reaction have greatly improved analytical capability over the last decade, achieving shorter analysis time with quantitative data and strain specificity, and in some cases the ability to discriminate cell viability. The emerging field of nanosensors/biosensors has demonstrated a variety of devices that hold promise to bridge the gap between traditional plate counting and molecular techniques. Many nanosensors/biosensors are rapid, portable, accurate devices that can be used as an additional screening tool for identifying unsafe levels of microorganisms in food products with no need for pre-enrichment. In this review, we provide a brief overview of available biorecognition-transduction techniques for detecting bacteria in food. We then discuss the advantages and disadvantages of each technique, and describe some recent biosensor or nanosensor technologies that are under development. We conclude by summarizing the opportunities and challenges in the field of pathogen monitoring in food systems and we focus the discussion on the strengths/weaknesses of the most popular biorecognition agents and transducer nanomaterials for biosensing.

Redefining the effect of salt on thermophilic starter cell viability, culturability and metabolic activity in cheese

This study investigated the differential effect of salt concentration in the outside and inside layers of brine salted cheeses on viability, culturability and enzyme activity of starter bacteria. The high-salt environment of the outside layer caused a sharp decrease in L. helveticus viability as measured by traditional plate counts. Remarkably, this was associated with lower release of intracellular enzymes (LDH), reduced levels of proteolysis and larger membrane integrity as measured by flow cytometry (FC) following classical Live/Dead staining. FC analysis of light scattering properties highlighted a significant reduction in size and granularity of the microbiota located in the cheese surface, suggestive of cell shrinkage and condensation of internal macromolecules probably due to hyperosmotic stress. The microbiota of the cheese surface were found to experience greater oxidative stress, as measured by FC analysis of the total levels of reactive oxygen species, compared to that of the interior layer. These results lead us to postulate that the physiology and health status of the microbiota were significantly different in the outer and inner layers of the cheese. The hyperosmotic environment of the outer layer resulted in reduced cell lysis, as measurable by assays based upon membrane integrity, but rather triggered cell death via mechanisms involving cell shrinkage and ROS-mediated damage of vital intracellular components. This study challenges the current thinking on how salt controls microbial activity in ripening cheese, especially in cheeses which are brine salted as local variations in biochemical ripening indices can differ significantly from the outside to the inside of a ripening cheese.

PH-Sensitive degradable polymeric micelles for bio-triggered targeted anti-tumor drug delivery

Sanitation is a critical step to reduce microbial load in many process industries. To assess and monitor the efficacy of sanitation processes, there is a need to rapidly measure microbial resistance to sanitizers and determine the critical concentration of sanitizers above which significant reduction in microbial load can be achieved in a complex environment. In this study, a rapid and label-free magnetic resonance (MR) imaging and relaxometry method was developed to measure resistance of Escherichia coli and Listeria monocytogenes to hydrogen peroxide based on the activity of intracellular catalase enzymes. Intracellular catalase activity is a measure of resistance of bacteria to hydrogen peroxide as these enzymes can degrade hydrogen peroxide to oxygen and water. Using sub-lethal and lethal concentration of hydrogen peroxide, the resistance of both the exponential and stationary growth phase E. coli suspended in PBS and in high organic content solution was characterized. The results show that in stationary growth phase E. coli, molecular oxygen, generated by the interaction of endogenous catalases with 1% H2O2, results in an 8 fold increase in MR imaging signal intensity and a 22 fold decrease in spin-lattice relaxation time (T1) compared to the controls. Furthermore, the resistance of E. coli and L. monocytogenes to H2O2, measured using the MR method, was correlated with the bacterial reduction, quantified with traditional plate counting method. The comparison between the two methods shows the MR method developed in this study can determine the critical concentration of H2O2 above which a significant reduction in bacterial culturability (at least 4 logs) was achieved. Overall, this study demonstrates the potential of MR imaging and relaxometry method to predict the efficacy of H2O2 in high organic load environment.

Effect of thyme essential oil and Lactococcus lactis CBM21 on the microbiota composition and quality of minimally processed lamb’s lettuce

The main aim of this work was to evaluate, at pilot scale in an industrial environment, the effects of the biocontrol agent Lactococcus lactis CBM21 and thyme essential oil compared to chlorine, used in the washing step of fresh-cut lamb’s lettuce, on the microbiota and its changes in relation to the time of storage. The modification of the microbial population was studied through pyrosequencing in addition to the traditional plate counts. In addition, the volatile molecule and sensory profiles were evaluated during the storage. The results showed no significant differences in terms of total aerobic mesophilic cell loads in relation to the washing solution adopted. However, the pyrosequencing data permitted to identify the genera and species able to dominate the spoilage associations over storage in relation to the treatment applied. Also, the analyses of the volatile molecule profiles of the samples during storage allowed the identification of specific molecules as markers of the spoilage for each different treatment. The sensory analyses after 3 and 5 days of storage showed the preference of the panelists for samples washed with the combination thyme EO and the biocontrol agent. These samples were preferred for attributes such as flavor, acceptability and overall quality. These results highlighted the effect of the innovative washing solutions on the quality of lettuce through the shift of microbiota towards genera and species with lower potential in decreasing the sensory properties of the product.

Fluorescent carbon dots with tunable negative charges for bio-imaging in bacterial viability assessment

Applying bio-imaging in bacterial viability assessment is vital to human health. Nitrogen, phosphorus co-doped carbon dots (NPCDs) with high visible fluorescence and tunable negative charges were synthesized by a feasible hydrothermal reaction. The as-prepared NPCDs possess abundant surface functional groups, excellent photostability and bio-compatibility. Especially, the surface negative charges of NPCDs can be adjusted by changing the kind of the precursors. The obtained NPCDs as a fluorescent dye could selectively stain dead bacteria instead of live ones due to electrostatic repulsion since the cell walls of bacteria possess negative charges. The NPCDs with less negative charges provides a low accurate mortality rate due to a weaker repulsion, while NPCDs with the most negative charges give a comparable mortality rate with traditional plate counting method but consumes shorter time. Our study suggests that NPCDs with highly negative charges could serve as a precise and efficient fluorescent probe to determine the living state of bacteria, widening the path for bio-imaging application in bacterial viability assessment.

Efficacy of benzalkonium chloride against bioluminescent P. aeruginosa ATCC9027 constructs

Whole cell biosensors have been seldom used in the pharmaceutical and cosmetics industries for preservative efficacy testing (PET). According to several pharmacopoeias, preservatives should be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. The aim of the study was to assess the applicability of Pseudomonas aeruginosa ATCC9027 and its validated bioluminescent strains for preservative efficacy tests using benzalkonium chloride (BKC). Applicability of five constitutively-expressed bioluminescent strains was evaluated for preservative efficacy tests (PET) using bacterial replication, bioluminescence and fluorescence in a three-way study. PET using BKC showed no significant difference between bioluminescence and enumeration. Good correlations between bioluminescence, colony-forming units (CFU) count and fluorescence were obtained for BKC concentrations (R>0.9) between 0.0003–0.0025% against strains containing the constructs lys-pMElux, lpp-pMElux and tat-pMElux. Furthermore, two-way ANOVA analysis showed that the bioluminescent method and traditional plate counting method were equivalent for concentrations of BKC (0.0003−0.01%) during preservative efficacy tests. PET testing with BKC showed that tat-pMElux (R>0.9) had consistently high correlation coefficients between CFU and relative bioluminescence; P. aeruginosa ATCC9027 tatH5-pMElux is the best construct for testing various antimicrobial agents.

A new application of a sodium deoxycholate-propidium monoazide-quantitative PCR assay for rapid and sensitive detection of viable Cronobacter sakazakii in powdered infant formula.

A rapid, reliable, and sensitive method for the detection of Cronobacter sakazakii, a common foodborne pathogen that may cause serious neonatal disease, has been developed. In this study, a rapid real-time quantitative PCR (qPCR) assay combined with sodium deoxycholate (SD) and propidium monoazide (PMA) was developed to detect C. sakazakii contamination in powdered infant formula (PIF). This method could eliminate the interference from dead or injured bacteria. Optimization studies indicated that SD and PMA at 0.08% (wt/vol) and 5µg/mL, respectively, were the most appropriate. In addition, qPCR, PMA-qPCR, SD-PMA-qPCR, and plate count assays were used to account for the number of viable bacteria in cell suspensions that were exposed to a 55°C water bath at different length of time. As a result, the viable number by PMA-qPCR showed significantly higher than of the number from SD-PMA-qPCR or plate counts. The number of viable bacteria was consistent between SD-PMA-qPCR and traditional plate counts, which indicated that SD treatment could eliminate the interference from dead or injured cells. Using the optimized parameters, the limit of detection with the SD-PMA-qPCR assay was 3.3×102 cfu/mL and 4.4×102 cfu/g in pure culture and in spiked PIF, respectively. A similar detection limit of 5.6×102 cfu/g was obtained in the presence of the Staphylococcus aureus (107 cfu/mL). The combined SD-PMA-qPCR assay holds promise for the rapid detection of viable C. sakazakii in PIF.

Performance Equivalence and Validation of the Soleris Automated System for Quantitative Microbial Content Testing Using Pure Suspension Cultures.

Using United States Pharmacopeia-National Formulary (USP-NF) general method <1223> guidance, the Soleris(®) automated system and reagents (Nonfermenting Total Viable Count for bacteria and Direct Yeast and Mold for yeast and mold) were validated, using a performance equivalence approach, as an alternative to plate counting for total microbial content analysis using five representative microbes: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans, and Aspergillus brasiliensis. Detection times (DTs) in the alternative automated system were linearly correlated to CFU/sample (R(2) = 0.94-0.97) with ≥70% accuracy per USP General Chapter <1223> guidance. The LOD and LOQ of the automated system were statistically similar to the traditional plate count method. This system was significantly more precise than plate counting (RSD 1.2-2.9% for DT, 7.8-40.6% for plate counts), was statistically comparable to plate counting with respect to variations in analyst, vial lots, and instruments, and was robust when variations in the operating detection thresholds (dTs; ±2 units) were used. The automated system produced accurate results, was more precise and less labor-intensive, and met or exceeded criteria for a valid alternative quantitative method, consistent with USP-NF general method <1223> guidance.

Assessment of sanitation efficacy against Escherichia coli O157:H7 by rapid measurement of intracellular oxidative stress, membrane damage or glucose active uptake

To reduce cross contamination with foodborne pathogens during washing procedures, sodium hypochlorite (NaOCl) and hydrogen peroxide (H2O2) are widely used in the fresh produce industry. To ensure food safety and minimize excessive use of sanitizers and water during sanitation, it is critical to develop a rapid method to assess sanitation efficacy. This study examines the potential for employing oxidative stress, membrane damage and glucose uptake measurements to assess the antimicrobial efficacy of NaOCl (0–70.4 ppm) and H2O2 (0–1.6% v/v) toward Escherichia coli O157:H7. The relative amount of intracellular reactive oxygen species (ROS) was measured using Aminophenyl fluorescein (APF) and 2′,7′- dichlorodihydrofluorescein diacetate (H2DCFDA) for cells treated with NaOCl and H2O2 respectively. Results from these ROS sensitive probes revealed a limited correlation between these oxidative stress measurements and inactivation of bacteria measured using the plate counting method. Sanitation experiments, conducted with contaminated pre-cut lettuce leaves in water, were also carried out. Measurement of the bacterial membrane integrity was assessed using the membrane permeable probe propidium iodide (PI) and by evaluating effects on active transport by the glucose transport system using 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG). The results demonstrate that the relative increase in membrane permeability using PI correlated with NaOCl, but did not correlate with H2O2 induced reduction of bacterial survival quantified by the plate counting method. In contrast to the other approaches, monitoring the loss of glucose transport system function by measuring the uptake of 2-NBDG displayed strong correlation with the reduction of bacterial survival for both NaOCl and H2O2 treatment. Overall, the study demonstrated potential of glucose uptake measurements with 2-NBDG to serve as surrogate method for the traditional plate counting, which is the current gold standard used by the Food and Drugs Administration, to provide a faster analysis of sanitation processes.

Ozone treatments of post harvested wine grapes: Impact on fermentative yeasts and wine chemical properties

Ozone represents a potent antimicrobial compound that is already proposed as a possible sanitizing agent, especially for surface decontamination of fruits and vegetables. The main objective of this study was to evaluate the effect of ozone, either in aqueous or gaseous form, on wine grape mycobiota and its impact during spontaneous and inoculated fermentations. Gaseous (32±1μL/L, 12 and 24h) and aqueous (5±0.25mg/L, 6 and 12min) ozone were tested as sanitizing treatments. A multiphasic approach was used employing culture-dependent (traditional plate counts) and -independent techniques, based on DNA and RNA amplification (PCR-denaturing gradient gel electrophoresis [DGGE] and reverse transcription PCR [RT-PCR]-DGGE), respectively. Microbiological analysis data highlighted a reduction of >0.5LogCFU/mL of the total yeasts present on grape berry surfaces after ozone treatments, mainly due to the reduction of apiculate yeasts. The chemical analysis of the wines, produced from the treated grapes, showed higher acetic acid content in the untreated spontaneous fermentations (0.52g/L) compared to the treated (ranged from 0.16 to 0.38g/L), while all fermentation-inoculated wines contained higher amounts of pleasant volatile compounds.

Behavior of Vibrio parahemolyticus cocktail including pathogenic and nonpathogenic strains on cooked shrimp

Pathogenic Vibrio parahaemolyticus, an important foodborne bacterium, coexists with nonpathogenic strains of V. parahaemolyticus in the environment. However, current predictive models for V. parahaemolyticus usually focused on single strain without considering the pathogenic and nonpathogenic bacteria cocktail coexist situation. In this study, the behavior of four V. parahaemolyticus strains (F18: tlh+/trh+/tdh−, ATCC 17802: tlh+/trh+/tdh−, F36: tlh+/trh−/tdh−, ATCC 33847: tlh+/trh−/tdh+) and their cocktails on cooked shrimp were investigated at different temperatures (4, 7, 10, 15, 20, 25 and 30 °C). Total bacteria counts were enumerated by traditional plate count method, periodically. Results showed both V. parahaemolyticus cocktails and four single strains grew in a temperature range of 15–30 °C and died at 4–7 °C. At 10 °C, for the cocktails, it inactivated at initial 100 h and then grew rapidly; but for single strain, only one strain F18 displayed the similar growth tendency, while the others grew all the time. Compared with single strains, the primary and secondary model analysis showed that the cocktails displayed a better growth activity with higher maximum growth rate and shorter lag time. The above results indicated that modeling bacterial growth by using a cocktail of V. parahaemolyticus strains may be a better option for simulating growth in the real environment. This study will fill in the gap of predictive microbiology and improve significantly the accuracy of microbial risk assessment.

Uncertainty of quantitative microbiological methods of pharmaceutical analysis

The total uncertainty of quantitative microbiological methods, used in pharmaceutical analysis, consists of several components. The analysis of the most important sources of the quantitative microbiological methods variability demonstrated no effect of culture media and plate-count techniques in the estimation of microbial count while the highly significant effect of other factors (type of microorganism, pharmaceutical product and individual reading and interpreting errors) was established. The most appropriate method of statistical analysis of such data was ANOVA which enabled not only the effect of individual factors to be estimated but also their interactions. Considering all the elements of uncertainty and combining them mathematically the combined relative uncertainty of the test results was estimated both for method of quantitative examination of non-sterile pharmaceuticals and microbial count technique without any product. These data did not exceed 35%, appropriated for a traditional plate count methods.

Combined Effect of Ultrasound and Ozone on Bacteria in Water.

The aim of this study is to assess the synergetic effect of combined ultrasound and ozone treatment on the biological disinfection of water on a large-scale application using viable plate counts and flow cytometry. Escherichia coli B bacteria in saline suspension was treated using a commercially available combined ultrasound and ozone system (USO3 (Ultrasonic Systems Gmbh)) for 16 min. Two analytical methods were used to assess the results in terms of live and dead cells in the bulk liquid: standard viable plate counting recorded in terms of colony forming units per milliliter and flow cytometry. In the latter case 1 mL of bacterial suspension was stained simultaneously with the fluorescent stains SYTO9 and propidium iodide (PI). Transmission electron microscopy was used to generate images identifying the biological effects of different treatments using ultrasound and ozone on bacterial cell walls. Results demonstrated that treatment with ozone alone (1 mg/L) resulted in a significant reduction (93%) in the number of live cells after 16 min treatment whereas ultrasound alone showed only a small reduction (24%). However, a combination of ozone and ultrasound showed a synergistic effect and enhanced the inactivation to 99% after 4 min. A combined ultrasound and ozone treatment of bacterial suspensions using a commercial system affords a promising method for water disinfection that is better than treatment using either method alone. Standard viable plate count analysis is normally used to assess the effectiveness of disinfection treatments; however flow cytometry proved to be a more sensitive method to determine the actual effects in terms of not only live and dead cells but also damaged cells. This type of analysis (cell damage) is difficult if not impossible to achieve using traditional plate counting methodology.

Photocatalytic and Antimicrobial Activity of Printed Hybrid Titania/Silica Layers.

Stable mixed dispersion of TiO2 and SiO2 was developed and hybrid TiO2/SiO2 layers were fabricated by the direct inkjet patterning technique. The prepared layers were tested for photoinduced hydrophilicity and their photocatalytic activity was tested using stearic acid and dichloroindophenol as model compounds. The antimicrobial activity of prepared layers was tested and evaluated by the traditional plate counting method according to ISO 27447:2009, using Escherichia coli CCM 3988. Material printing proved to be well suited for the deposition of this complex nanoparticulate ink and samples with variable thickness were conveniently fabricated. Printed layers are able to change their surface properties from hydrophobic to superhydrophilic and also decompose the model contaminants rapidly.