Membrane Integrity Test Research Articles

Effects of high hydrostatic pressure on Escherichia coli ultrastructure, membrane integrity and molecular composition as assessed by FTIR spectroscopy and microscopic imaging techniques.

High hydrostatic pressure (HHP) is a novel food processing technology that is considered as an attractive alternative to conventional heat treatments for the preservation of foods, due to its lethal effects on pathogenic and spoilage microorganisms, while causing minor effects on food quality and sensorial attributes. This study is aimed at investigating how HHP treatments at varying intensities in the range 50–900 MPa affect the viability, membrane integrity, ultrastructure and molecular composition of Escherichia coli. Results of membrane integrity tests (measurement of cellular leakage and monitoring of propidium iodide uptake through fluorescence microscopy) and ultrastructural observations by transmission electron microscopy demonstrated that HHP gave rise to cellular enlargement, membrane damage or detachment, DNA and protein denaturation and loss of intracellular contents. Fourier-transform infrared (FTIR) spectroscopy analyses evidenced minor changes in molecular composition in response to high pressures, which were mostly observed on the spectral region w4 (1200–900 cm−1), mainly informative of carbohydrates and polysaccharides of the cell wall. These findings suggest that exposure of E. coli cells to HHP causes alterations in their physical integrity while producing minor modifications in biochemical cellular composition. The current study increases the knowledge on the mechanisms of E. coli inactivation by HHP and provides valuable information for the design of more effective food preservation regimes based on the integration of mild HHP in combination with other food preservation strategies into a multi-target hurdle technology approach.

Analysis of performance criteria for ultrafiltration membrane integrity test using magnetic nanoparticles

An alternative ultrafiltration membrane integrity test utilizing magnetic nanoparticle as a surrogate has been investigated in previous studies, but the absence of a feasible estimation model for the degree of membrane damage causes that this simple membrane integrity test would be not applied easily. This study proposed a calculating model to predict membrane defect size, and investigated the theoretic resolution of the integrity test method. The results obtained with the evolved prediction model D, which is based on Darcy’s law and Bernoulli equation, were satisfactory in predicting the membrane defect size. In this study, this integrity test method had about 39.33% probability to have a theoretic resolution of 3μm or less under common experimental conditions.

AVALIAÇÃO DA EFICÁCIA DE CRIOPROTETORES PERMEANTES E NÃO PERMEANTES NO DESCONGELAMENTO RÁPIDO E LENTO DO SÊMEN CANINO

The aim of this study was to evaluate the association between cryoprotectants little studied in Brazil such as dimethylformamide and trehalose amid thinner, using protocols of fast and slow defrosting. Three adult Labrador Retrievier male, healthy dogs, weekly submitted to one semen collection during five-weeks period, were used. The base diluent medium used in this study was tris-citrate added with 3% of dimethylformamide + 3% glycerol (D1), 3% dimethylformamide and trehalose (D2) and 4% glycerol (D3). At defrosting, half of the semen samples from each diluent medium was defrosted by rapid method in water-bath at 75 °C for seven minutes, followed by a new immersion at 37 °C for 1 minute. The other half of the samples was defrosted by slow method, in water-bath at 37 °C for 1 minute. The semen was evaluated for sperm progressive motility and vigor, besides membrane integrity. For this, the semen samples were submitted to either hyposmotic and membrane integrity tests of the plasmatic membrane and acrosome (fluorescence). The results indicated that the use of glycerol as cryoprotector in TRIS diluter provides greater efficacy in cryopreserving spermatozoa of the canine species, when compared to dimethylformamide associated with trehalose or glycerol.KEYWORDS: cryopreservation; dimethylformamide; dogs; glycerol; trehalose.

Fluorescent Fingerprints of Endolithic Phototrophic Cyanobacteria Living within Halite Rocks in the Atacama Desert

Halite deposits from the hyperarid zone of the Atacama Desert reveal the presence of endolithic microbial colonization dominated by cyanobacteria associated with heterotrophic bacteria and archaea. Using the λ-scan confocal laser scanning microscopy (CLSM) option, this study examines the autofluorescence emission spectra produced by single cyanobacterial cells found inside halite rocks and by their photosynthetic pigments. Photosynthetic pigments could be identified according to the shapes of the emission spectra and wavelengths of fluorescence peaks. According to their fluorescence fingerprints, three groups of cyanobacterial cells were identified within this natural extreme microhabitat: (i) cells producing a single fluorescence peak corresponding to the emission range of phycobiliproteins and chlorophyll a, (ii) cells producing two fluorescence peaks within the red and green signal ranges, and (iii) cells emitting only low-intensity fluorescence within the nonspecific green fluorescence signal range. Photosynthetic pigment fingerprints emerged as indicators of the preservation state or viability of the cells. These observations were supported by a cell plasma membrane integrity test based on Sytox Green DNA staining and by transmission electron microscopy ultrastructural observations of cyanobacterial cells.

Antibacterial activity and mode of action of a commercial citrus fruit extract

This study addresses the antibacterial activity and mechanism of action of BIOLL(+®), a commercial extract obtained from citrus fruits. Strong activities with minimum inhibitory concentrations (MIC) ranging from 10 ppm (for some Brachyspira hyodysenteriae strains) to 80 ppm (for various Salmonella enterica and Escherichia coli strains) were observed. Membrane integrity tests and Fourier transform infrared (FT-IR) spectroscopic analyses were performed to shed light on the effects caused on molecular structure and composition. Physical effects, with formation of pores and leakage of intracellular components, and chemical effects, which were dependent on the bacterial species, were evident on cellular envelopes. Whereas for S. enterica and E. coli, changes were focused on the carboxylic group of membrane fatty acids, for B. hyodysenteriae, the main effects were found in polysaccharides and carbohydrates of the cell wall. The great antibacterial activity shown by BIOLL(+®) and its proposed dual physico-chemical mode of action, with species-specific cellular targets, show its attractiveness as an alternative to antibiotics. Antibiotic resistance is becoming a serious problem. Our study characterizes a novel antimicrobial extract, which could represent an alternative to antibiotics for treatment or prevention of bacterial infectious diseases.

Cassia oil as a substitute solvent for xylene for rotenone EC and its synergistic activities

The purpose of this study was to screen essential oils as substitute solvents for rotenone emulsifiable concentrates (EC). Spodoptera litura (SL-1) cell and third-instar larvae of S. litura were used as the model system to evaluate the cytotoxicity of essential oils and synergistic effect of cassia oil with rotenone. The result of screening assay indicated that essential oil treatments led to reductions in cell viability in a time-dependent manner. The inhibition rates of the 15 essential oils were over 80% after SL-1 cells were treated with 200μg/mL essential oils for 48h. The solubility of rotenone in cassia, basil, and clove oils were 15.84, 6.86, and 5.48g/100g, respectively, much higher than their solubility in xylene. After 24h treatment, the rotenone and cassia oil mixture (1:35, w/w) featured half maximal inhibitory concentration (IC50) of 28.66μg/mL against SL-1 cells and median lethal concentration (LC50) of 513.33μg/mL against third-instar larvae of S. litura. The co-toxicity coefficients(CTCs) of rotenone and cassia oil mixture (1:35, w/w) against SL-1 cells and third-instar larvae of S. litura were 240.75 and 166.23 when treated for 24h, which indicated that cassia oil possessed good synergistic effect with rotenone. The cell membrane potential and membrane integrity test results showed that cassia oil induced potential hyperpolarization of SL-1 cell membranes and promoted accumulation of PI, indicated that oil-exposed membranes lose cell integrity. Morphological images showed that application of rotenone with cassia oil to SL-1 cells produced cell membranes with their contents leaking. Finally, high-performance liquid chromatography (HPLC) assays proved that cassia oil increases the absorption of rotenone. These findings confirmed that cassia oil has marked synergistic effect on rotenone and has strong potential as an alternative solvent for rotenone EC.

What Should I Know About Membrane Integrity Testing to Comply With LT2ESWTR?

The U.S. Environmental Protection Agency (USEPA) published the Long‐Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR) more than six years ago. To comply with the rule, many utilities have chosen low‐pressure membranes ‐ microfiltration and ultrafiltration ‐ which are effective for pathogen removal as long as membrane integrity is intact. However, membrane integrity must be verified. This article sums up the basics of membrane integrity testing in 10 questions and answers.

Video analysis of osmotic cell response during cryopreservation

Cellular response during the freeze–thaw process strongly affects the cryopreservation outcome including cell morphology and cell viability. Cryomicroscopy was used to individually analyze the osmotic response of human pulmonary microvascular endothelial cells (HPMECs) during slow cooling (1°C/min) to −60°C and fast rewarming to 4°C (100°C/min). The ice nucleation temperature was controlled (Tn=−8°C). Different concentrations of different cryoprotectant agents, dimethyl sulfoxide, ethylene glycol, proline, ectoin, and trehalose resulted in various cell volume changes. The described methods for image processing and computer vision allows for a fully automatic and individual analysis of the osmotically driven cell response under a temporal resolution of 2frames per second. As a result, we show that in the presence of dimethyl sulfoxide or ethylene glycol cells shrink during cooling to a high degree, especially at intermediate molar concentrations in the range between 0 and 2M, while during rewarming cells swell to isotonic volumes gradually. Comparative cell vitality tests, membrane integrity, and viability tests after 24h recultivation, under these conditions show a high cell survival. In the absence of cryoprotective agents or with proline, ectoin or trehalose, osmotic shrinkage did not meet our expectations: a freeze-induced swelling was detected during cooling and an extreme swelling was observed after rewarming, which was accompanied by lower comparative cell viability. A linear correlation between the cellular membrane integrity after cryopreservation and the maximal relative cell volume was derived (R2=96). The results clearly show that it is crucial to analyze cells within a sample individually due to their individual different osmotic response.

Application of genetic programming to develop the model for estimating membrane damage in the membrane integrity test using fluorescent nanoparticle

A new approach utilizing silica fluorescent nanoparticle as a surrogate for checking the integrity of microfiltration membrane was proposed and well applied in a previous study, but the absence of a feasible estimation model for the degree of membrane damage caused that this simple membrane integrity test was not applied easily. This study proposes genetic programming (GP) as an alternative approach to develop the model to predict the area of membrane breach with other experimental conditions (concentration of fluorescent nanoparticle, the permeate water flux and transmembrane pressure). Unlike the artificial neural network that is the most common artificial intelligence technique, GP is an inductive data-driven machine learning that evolves an explicit equation with known experimental data. The results obtained with GP models evolved were satisfactory in predicting the area of the membrane breach and, with the simple membrane integrity test, the GP technique gives a practical way for estimating the degree of membrane damage. Therefore, GP could serve as a robust approach to develop an estimation model for the new membrane integrity test.

Understanding the impact of divalent cation substitution on hydroxyapatite: An in vitro multiparametric study on biocompatibility

Hydroxyapatite (HA), a stable and biocompatible material for bone tissue therapy, may present a variable stoichiometry and accept a large number of cationic substitutions. Such substitutions may modify the chemical activity of HA surface, with possible impact on biocompatibility. In this work, we assessed the effects of calcium substitution with diverse divalent cations (Pb(2+), Sr(2+), Co(2+), Zn(2+), Fe(2+), Cu(2+), or Mg(2+)) on the biological behavior of HA. Physicochemical analyses revealed that apatite characteristics related to crystallinity and calcium dissolution/uptake rates are very sensitive to the nature of cationic substitution. Cytocompatibility was evaluated by mitochondrial activity, membrane integrity, cell density, proapoptotic potential, and adhesion tests. With the exception of Zn-HA, all the substituted HAs induced some level of apoptosis. The highest apoptosis levels were observed for Mg-HA and Co-HA. Cu-HA was the only material to impair simultaneously mitochondrial activity, membrane integrity, and cell density. The highest relative cell densities after exposure to the modified HAs were observed for Mg-HA and Zn-HA, while Co-HA significantly improved cell adhesion onto HA surface. These results show that changes on surface dissolution caused by cationic substitution, as well as the increase of metal species released to biological media, were the main responsible factors related to alterations on HA biocompatibility.

Water purification from pesticides by spiral wound nanofiltration membrane

An alternative ultrafiltration membrane integrity test was already developed in laboratory scale. It is based on the use of magnetic nanoparticles (Fe3O4) and measurement of magnetic susceptibility. The mean size of nanoparticles used is around 35 nm and they show a good disparity between 20 and 100 nm. In this paper, validation of this membrane integrity monitoring method was achieved by industrialscale tests. Two holes with 0.6 mm internal diameter in a module containing 10 000 fibers (35 m2 surface area) was efficiently detected by injecting 750 mL of 1.7 g.L−1 nanoparticle solution during 2s when the test was operated at low TMP (0.096 bar, corresponding to a flux of 2.2 m3.h−1). In addition, it has been demonstrated that within the detectable range, this membrane integrity test with magnetic nanoparticles has a very rapid response time. The response time depends on the permeate flux and the dead-volume of the pilot. This membrane integrity test, with the advantages of on-line operation, high detection sensitivity, detection specificity and very low influence on membrane fouling, seems to be suitable for large scale drinking water plants.

Use of fluorescent silica particles for checking the integrity of microfiltration membranes

In this study, a new approach utilizing silica-based fluorescent particles as a surrogate for checking the integrity of microfiltration membranes was systematically investigated. The fluorescent particles were synthesized as spheres with diameters of 0.5–0.7 μm and used to test the integrity of microfiltration membranes with nominal pore sizes of 0.25 μm. The fluorescent particles only flowed through the damaged parts of membranes. The permeates were then collected and filtered through a GF/C filter to collect the outflow particles. The collected particles were placed under a UV lamp, and the fluorescence was photographed with a digital camera. The mass of the outflow particles was estimated by analyzing the images of the fluorescence pictures. To estimate the size of damage on the membrane surface, the functional relationship between the damaged area of the membrane and the mass of fluorescent outflow particles was established using a dimensional analysis. In addition, the dead-end filtration results show that at particle concentrations >10 mg/L, the flux decline was approximately 10% even then the particle concentration was increased to 100 mg/L. The cake resistance caused by particle deposition increased the total membrane resistance only by 10% in long-term filtration. Therefore, the synthesized fluorescent silica particles are a suitable surrogate for membrane integrity tests.

Changes in ultrastructure and Fourier transform infrared spectrum of Salmonella enterica serovar typhimurium cells after exposure to stress conditions.

The effect of exposure to acid (pH 2.5), alkaline (pH 11.0), heat (55°C), and oxidative (40 mM H₂O₂) lethal conditions on the ultrastructure and global chemical composition of Salmonella enterica serovar Typhimurium CECT 443 cells was studied using transmission electron microscopy and Fourier transform infrared spectroscopy (FT-IR) combined with multivariate statistical methods (hierarchical cluster analysis and factor analysis). Infrared spectra exhibited marked differences in the five spectral regions for all conditions tested compared to those of nontreated control cells, which suggests the existence of a complex bacterial stress response in which modifications in a wide variety of cellular compounds are involved. The visible spectral changes observed in all of the spectral regions, together with ultrastructural changes observed by transmission electron microscopy and data obtained from membrane integrity tests, indicate the existence of membrane damage or alterations in membrane composition after heat, acid, alkaline, and oxidative treatments. Results obtained in this study indicate the potential of FT-IR spectroscopy to discriminate between intact and injured bacterial cells and between treatment technologies, and they show the adequacy of this technique to study the molecular aspects of bacterial stress response.

Application of magnetic nanoparticles for UF membrane integrity monitoring at low-pressure operation

Abstract An alternative ultrafiltration membrane integrity test is presented and evaluated, based on the use of magnetic nanoparticles (Fe 3 O 4 ) and measurement of magnetic susceptibility. The mean size of nanoparticles used is around 35 nm and they show a good disparity between 20 and 100 nm. A series of integrity tests were carried out on a Norit membrane module containing 100 fibers under a low transmembrane pressure of 0.25 bar. The results showed that no magnetic susceptibility was detected in permeate when the tests were performed on the intact module in both cross-flow and dead-end filtration, indicating the complete nanoparticle retention by the intact module. However, when even one fiber was broken in the module (1% breakage rate), magnetic susceptibility of permeate could be detected instantaneously even at feed concentrations as low as 1.2 ppm with Bartington magnetic susceptibility meter. This detection is valid during all the filtration process. The results also showed that the membrane permeability could be completely recovered after a backwash. This membrane integrity test, with the advantages of simplicity, on-line operation, high detection specificity and sensitivity, quick detection and very low influence on membrane fouling, seems to be suitable for large-scale drinking water plants.

Use of pressure-hold test for sterilizing filter membrane integrity in radiopharmaceutical manufacturing

The bubble point test is the de facto standard for postproduction filter membrane integrity test in the radiopharmaceutical community. However, the bubble point test depends on a subjective visual assessment of bubbling rate that can be obscured by significant diffusive gas flows below the manufacturer's prescribed bubble point. To provide a more objective means to assess filter membrane integrity, this study evaluates the pressure-hold test as an alternative to the bubble point test. In our application of the pressure-hold test, the nonsterile side of the sterilizing filter is pressurized to 85% of the predetermined bubble point with nitrogen, the filter system is closed off from the pressurizing gas and the pressure is monitored over a prescribed time interval. The drop in pressure, which has a known relationship with diffusive gas flow, is used as a quantitative measure of membrane integrity. Characterization of the gas flow vs. pressure relationship of each filter/solution combination provides an objective and quantitative means for defining a critical value of pressure drop over which the membrane is indicated to be nonintegral. The method is applied to sterilizing filter integrity testing associated with the commonly produced radiopharmaceuticals, [(18)F]FDG and [(11)C]PIB. The method is shown to be robust, practical and amenable to automation in radiopharmaceutical manufacturing environments (e.g., hot cells).

Improvement of the resolution in direct membrane integrity test by controlling solution surface tension

Membrane-based treatment technologies have been introduced as a promising tool for the removal of water-borne pathogens. To ensure successful application of membrane processes, the integrity of the membrane system should be maintained. Related with evaluation of the membrane integrity, USEPA guidance recommends pressure-based membrane integrity (MIT). Based on the bubble point theory, the ability of detecting smallest integrity breakage during the MIT is defined as "Resolution". However, the response to remarkably small breach demands significantly high initial test pressure of the pressure decay test. In this study, the surface tension of the test liquid was controlled to improve the resolution without increasing the corresponding test pressure. Three common chemicals were chosen to control the solution surface tension. It is concluded that 0.1 M of the citric acid can decrease the initial test pressure significantly for the same pore size. Subsequently, the improvement of the resolution with controlled surface tension was confirmed by the results of pressure decay test and marker test.

Use of paramagnetic particles in membrane integrity testing

Current online methods of testing membrane integrity – turbidity and particle counting/monitoring – are non-specific and offer moderate sensitivity. An alternative method of probing membrane integrity utilizing magnetically susceptible particles was evaluated as a means to improve both specificity and sensitivity. Compromised 0.6 μm polycarbonate track-etched membranes were challenged with magnetically susceptible particles of 1 μm diameter. Particles that breached the compromised membranes were successfully separated from the filtrate using a particle collector under the influence of a magnetic field. The particle capture efficiency decreased with higher filtrate flow velocity. The captured particles were readily released in the absence of a magnetic field allowing further concentration in a subsequent elution step and increased system sensitivity. Sample calculations show that the proposed technique is a plausible one for large-scale applications.

Arginine magic: Arginine kills tumor cells when presented in saline

There has been no anti‐cancer formulation so far, which is sufficiently effective for the treatment of cancer using an endogenous molecule. Various scientists have attempted to describe the complex role of arginine in cancer biology and treatment. Nobody has yet looked at the non‐substrate actions of arginine, to act as a Biological Response Modifier through its chemical structure. Arginine containing peptides have been used as delivery vectors to carry payloads of drugs. The results indicate that guanidinium group is critical for the translocation process. Based on the above, we hypothesized that pharmacological doses of L‐arginine (LA) when delivered in saline could interact with tumour cells more efficiently.Various tumor cell lines derived from mouse and human when incubated with LA, D‐arginine (DA) in pharmacological doses in PBS, caused the death of tumor cells, as assessed by the membrane integrity test, trypan dye exclusion test and MTT assay. There was complete destruction of cell structure (Fig.1). DA had better anticancer ability as it could inhibit the tumor growth at lower concentration. The anticancer property of LA is not accompanied by any toxicity to the red blood cells or NIH 3T3 cells. L‐lysine and agmatine did not offer any antitumor activity. Arginine administered in PBS could also cure the mice of the tumor transplanted into the peritoneum (Fig. 2). These findings provide a previously unrecognized beneficial effect of guanidinium group containing compounds in killing the tumor cells and thus provide new potential approaches for therapeutic development of arginine in tumor therapy.

Review of 18F-FDG synthesis and quality control

This review article covers a concise account on fludeoxyglucose (18F–FDG) synthesis and quality control procedures with emphasis on practical synthesis Currently, 18F–FDG is the most successful PET radiopharmaceutical so far. The advancement in synthesis and quality control of 18F–FDG, together with its approval by the US FDA and the availability of reimbursement, are probably the main reasons for the flourish of clinical PET over the last 20 years. 18F–FDG can be synthesised by either electrophilic fluorination or nucleophilic fluorination reaction. Nucleophilic fluorination using mannose triflate as precursor and Kryptofix or tetrabutylammonium salts (TBA) is widely used because of higher yield and shorter reaction time. The quality control requirements of 18F–FDG can be found in United States Pharmacopeia (USP), British Pharmacopeia (BP), European Pharmacopeia (EP) and the Chemistry, Manufacturing, and Controls (CMC) section from United States Food and Drug Administration (US FDA) PET draft guidance documents. Basic requirements include radionuclidic identity, radiochemical purity, chemical purity, pH, residual solvent, sterility, and bacterial endotoxin level. Some of these tests (sterility, endotoxins and radionuclidic purity) can be finished after the 18F–FDG has been released. Although USP, BP and EP do not require filter membrane integrity test, many laboratories perform this test as an indirect evident of the product sterility. It is also interesting to note that there are major differences in 18F–FDG quality requirements among USP, BP, and CMC.

Membrane filtration for cold regions – impact of cold water on membrane integrity monitoring tests

Low-pressure water treatment membranes, microfiltration (MF) and ultrafiltration (UF) have enjoyed unprecedented growth during the past decade. These systems are particularly suited for cold region communities because of their operational simplicity, small footprint, and consistent performance despite the fluctuations in raw water characteristics. Effective operation of MF and UF membrane systems in cold regions, however, must account for the impact of low water temperatures on membrane operation. Of particular interest is the impact of cold water on direct membrane integrity monitoring tests. Most regulatory bodies require that regular integrity monitoring tests be performed for low-pressure membranes to ensure the integrity of the membrane systems. Currently, the most widely-used integrity monitoring test is the pressure decay test. Our studies, however, indicated that the pressure-based direct integrity monitoring tests are affected by variations in water temperature, especially in the range of 0 to 5 °C. The considerable drop in diffusive air flow rates and consequent decrease in pressure decay rate for an intact membrane as water temperatures approach 0 may mask the impact of a defect and should be accounted for. It is suggested that the criteria for membrane integrity tests in cold regions shift downward to account for the effect of temperature. This paper presents the results obtained from studies on the effect of water temperature on the pressure-based direct integrity monitoring tests. Key words: membrane integrity monitoring, pressure decay test, diffusive air flow test, low-pressure membranes, microfiltration, ultrafiltration, water treatment, pathogen removal.