Conductimetric Assay Research Articles

Heated scallop-shell powder treatment for deactivation and removal of Listeria sp. biofilm formed at a low temperature.

The ability of heated scallop-shell powder (HSSP) to work against Listeria sp. biofilm formed at a low temperature was investigated. A biofilm of L. innocua ATCC 33090 was grown on a glass plate at 15˚C for 15 days, then immersed in HSSP slurry. Following treatment, the disinfection ability of the HSSP against the biofilm was non-destructively quantified by conductimetric assay. The biofilm grown at 15˚C was less sensitive than that grown at 37˚C to HSSP treatment and alkaline treatment. The biofilm grown at 15˚C was completely deactivated by 30 min of HSSP treatment (10 mg/mL, pH 12.5). In contrast, after 30 min treatment with alkaline solution at pH 12.5 or sodium hypochlorite (100 ppm), the activity was reduced by only one order of magnitude. The disinfection efficacy of HSSP (10 mg/mL) against L. innocua is similar to or higher than that of sodium hypochlorite (200 ppm). Fluorescence microscopy validated the results of the conductimetric assay. Therefore, HSSP treatment is a potentially powerful alternative control agent against Listeria sp. biofilms that present hazards in the food industry.

Growth inhibitory effect on bacteria of chitosan membranes regulated with deacetylation degree

Antibacterial activity of chitosan membranes was investigated by a conductimetric assay using a Bactometer. The purpose of this investigation was to produce a practical, high-performance membrane for separation engineering. The antibacterial activity of powdered chitosan membrane was evaluated by the minimal inhibitory concentration (MIC). The MIC for Escherichia coli was almost 200 (mg-chitosan/ml-bacterial suspension), and for Staphylococcus aureus it was 40 (mg-chitosan/ml-bacterial suspension). Growth of the gram-positive sample ( S. aureus) was more strongly inhibited by chitosan than the gram-negative sample ( E. coli). This inhibitory effect was recognized as a bactericidal effect. Antibacterial activity was also observed and depended on the shape and the specific surface area of the powdered chitosan membrane. The influence of the deacetylation degree (DD) of the chitosan on inhibiting the growth of S. aureus was investigated by two methods: incubation using a mannitol salt agar medium, and a conductimetric assay. By both methods, chitosan with a higher DD successfully inhibited growth of S. aureus. Our findings regarding the dominant role of the DD of chitosan will be useful for designing long-life, hygienic, membrane-based processes.

Antibacterial Activity of Carbon-Coated Zinc Oxide Particles

Particles of ZnO coated with carbon (ZnOCC) were prepared and evaluated for their antibacterial activity. ZnO powder and poly(vinyl alcohol) (PVA) (polymerization degree: 2,000-95,000) were mixed at a mass ratio (ZnO/PVA) of 1, and then heated at 500-650 degree C for 3 h under argon gas with a flow rate of 50ml/min. Carbon deposited on the ZnOCC surface was amorphous as revealed by X-ray diffraction studies. The ZnOCC particles maintained their shape in water, even under agitation. The antibacterial activity of ZnOCC powder against Staphylococcus aureus was evaluated quantitatively by measuring the change in the electrical conductivity of the growth medium caused by bacterial metabolism (conductimetric assay). The conductivity curves obtained were analyzed using the growth inhibition kinetic model proposed by Takahashi for calorimetric evaluation, allowing the estimation of the antibacterial efficacy and kinetic parameters of ZnOCC. In a previous study, when ZnO was immobilized on materials, such as activated carbon, the amount of ZnO immobilized was approximately 10-50%, and the antibacterial activity markedly decreased compared to that of the original ZnO. On the other hand, the ZnOCC particles prepared in this study contained approximately 95% ZnO and possessed antibacterial activity similar to that of pure ZnO. The carbon-coating treatment could maintain the antibacterial efficacy of the ZnO and may be useful in the develop-ment of multifunctional antimicrobial materials.

Quantitative evaluation of antifungal activity of metallic oxide powders (MgO, CaO and ZnO) by an indirect conductimetric assay.

To evaluate antifungal activities of MgO, CaO and ZnO powders quantitatively by indirect conductimetric assay. Candida albicans NBRC1060, Saccharomyces cerevisiae NBRC1950, Aspergillus niger NBRC4067 and Rhizopus stolonifer NBRC4781 were used as test micro-organisms. The indirect conductimetric assay, in which the change in electrical conductivity of an alkaline solution (NaOH) is produced by absorption of CO2 from microbial metabolism, could offer a simple and rapid evaluation of the antifungal activity within 24-48 h. The conductivity curves obtained for MgO, CaO and ZnO were analysed using the growth inhibition kinetic model proposed by Takahashi for calorimetric evaluation, and the kinetic parameters and minimum inhibitory concentration ([I]100) could be determined. MgO and CaO powders exhibited the antimicrobial activities against all fungi used in this study and showed little differences between types of fungi. However, although ZnO powder inhibited fungal growth, the values of [I]100 were over 100 mg ml-1. Although a common method for evaluating antifungal activity requires over 5-7 days, the indirect assay could provide a rapid and quantitative evaluation of antifungal activity within approx. 2 days, and MgO and CaO were found to have antifungal activities. The indirect assay can be applicable for simple and rapid evaluation of the antimicrobial activity of insoluble or slightly soluble materials with high turbidity such as antibacterial ceramic powders. Moreover, these materials can be useful for controlling fungi in food processing and the environment.

Decrease in Toxic Potential of Mixed Tensides Maintained below the Critical Micelle Concentration: An in vitro Study

Sodium lauryl sulphate (SLS) is an anionic tenside widely utilized in commercial topical preparations that may cause skin irritation. It has been shown that the barrier damage caused by SLS in vivo is lower when SLS is used in combination with other tensides which are able to reduce the critical micelle concentration (CMC). The aim of our study was to evaluate if the cytotoxic effect of SLS is reduced by the association with different tensides also at concentrations well below the CMC. Normal human keratinocytes from plastic surgery were grown in serum-free medium. At subconfluency, the cells were treated with SLS at a dose of 0.0025% in combination with cocamidopropyl betaine, Tween 20 and Tween 80 at the minimum toxic dose. Following tenside treatment, the culture medium was changed, and after 24 h the cells were collected for ³H-thymidine incorporation, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and neutral red (NR) uptake. The cytotoxic effect on normal human keratinocytes, as evaluated by ³H-thymidine incorporation, MTT assay and NR uptake, was significantly decreased by the combination with all the tested tensides. The correlation between cytotoxicity and physical properties was also studied by a conductimetric assay to investigate the mechanism involved in this toxicity reduction.

Quantitative evaluation of antibacterial activities of metallic oxide powders (ZnO, MgO and CaO) by conductimetric assay

Antibacterial activities of metallic oxide (ZnO, MgO and CaO) powders against Staphylococcus aureus and Escherichia coli were quantitatively evaluated by measuring the change in electrical conductivity of the growth medium caused by bacterial metabolism (conductimetric assay). The obtained conductivity curves were analyzed using the growth inhibition kinetic model proposed by Takahashi for calorimetric evaluation, and the metallic oxides were determined for the antibacterial efficacy and kinetic parameters. The parameters provide some useful indicators for antimicrobial agents, such as the dependence of antibacterial activity on agent concentration, and the affinity between the agent and the bacterial cells. CaO was the most effective, followed by MgO and ZnO, against E. coli. On the other hand, ZnO was the most effective for S. aureus and was suggested to have a strong affinity to the cells of S. aureus.

Indirect conductimetric assay of antibacterial activities

The applicability of indirect conductimetric assays for evaluation of antibacterial activity was examined. The minimal inhibitory concentration (MIC) obtained by the indirect method was consistent with that by the direct conductimetric assay and the turbidity method. The indirect assay allows use of growth media, which cannot be used in the direct conductimetric assay, making it possible to evaluate the antibacterial activity of insoluble or slightly soluble materials with high turbidity, such as antibacterial ceramic powders.

Indirect conductimetric assay of antibacterial activities.

The applicability of indirect conductimetric assays for evaluation of antibacterial activity was examined. The minimal inhibitory concentration (MIC) obtained by the indirect method was consistent with that by the direct conductimetric assay and the turbidity method. The indirect assay allows use of growth media, which cannot be used in the direct conductimetric assay, making it possible to evaluate the antibacterial activity of insoluble or slightly soluble materials with high turbidity, such as antibacterial ceramic powders.

Correlation of the Initial bacterial concentration and detection time in indirect conductimetric assay.

The correlation between bacterial concentration and the change in electrical conductivity was investigated for the indirect conductimetric assay which measures the conductivity change of an alkaline solution (NaOH) that absorbs the CO2 produced by bacterial growth and metabolism. The conductivity of the NaOH solution for a sample containing a higher bacterial concentration changed faster than that for a sample containing a lower concentration, and the logarithmic initial bacterial concentration (CB0) was related to the time (tD), at which the conductivity of the absorbent changed, in a linear fashion. We derived theoretically the validity of the linear relationship and estimated the generation time (tg) from the relationship. The tg values obtained by the indirect conductimetric assay were almost identical to those obtained by the direct conductimetric assay and the Standard Plate Count method.

Demonstration of a protein synthesis in starved Campylobacter jejuni cells

For many years, environmental microbiologists working on water samples, have reported differences between bacterial counts performed by culture and by microscopy. These observations have led to the demonstration of the viable but non-culturable (VNC) state in bacteria. Some hygienist specialists underlined the risk presented by pathogenic bacteria in the VNC state. The VNC state in bacteria has been studied by a number of authors, but the relation between VNC state and bacterial stress response has not been established yet, while the VNC state is generally described in responses to adverse conditions. Campylobacter jejuni enter the VNC state in response to starvation. In our study, we searched for a protein synthesis in the first hours of the cell starvation exposure. Three Campylobacter jejuni strains were suspended in filtered, sterilized, distilled water, and incubated at 4°C with gentle shaking (100 rpm). After 1, 2, 3, 4 and 5 h of starvation, C. jejuni cells were removed and subjected to a heat shock (55°C, 3 min) and to a conductimetric assay. Results obtained showed that a protein synthesis occurred in the onset of the starvation period, and that these improved the nutrient assimilation and enhanced the heat resistance in starved cells.

Analysis of conductance responses during depolymerization of pectate by soft rot Erwinia spp. and other pectolytic bacteria isolated from potato tubers.

Different bacteria isolated from potato tubers were screened for their pectolytic properties by examining pitting in polypectate agar, recording conductance responses in polypectate medium and performing potato tuber soft rot tests. For bacteria found positive in conductimetry, the role of polygalacturonase (PG) and pectate lyase (PL) in the generation of conductance changes in a polygalacturonic acid (PGA) medium was further analysed using enzyme activity staining after gel electrophoresis and high-performance anion exchange chromatography. The extent of the conductance changes during depolymerization of PGA was dependent on the amounts of galacturonate monomers and oligomers accumulated in the medium. In comparison with an unidentified saprophyte and a Klebsiella strain, both mainly having PL activity, soft rot Erwinia spp. rapidly produced larger conductance responses, due to a combined action of multiple forms of PG and PL. The responses of Erwinia spp. were initially associated with the accumulation of large amounts of monomers and saturated dimers to heptamers, due to PG activity. Subsequently, as well as monomers and saturated dimers, large amounts of unsaturated dimers were also detected, due to PL activity. The role of PG as an important conductimetric factor was also demonstrated for a pectinase preparation derived from Aspergillus niger. Besides detection, automated conductimetric assays in pectate media may also be useful for monitoring of pectolytic activity in pectinase preparations and for screening of pectolytic activity of microorganisms under different media and growth conditions.

The development of a conductimetric assay for automated detection of metabolically active soft rot Erwinia spp. in potato tuber peel extracts

Four media were tested for their ability to detect the soft rot potato pathogens Erwinia chrysanthemi (Ech) and Erwinia carotovora ssp. atroseptica (Eca) in potato tubers by means of automated conductance measurements. The specificity of the conductimetric assays was determined by testing a set of different Erwinia spp. and potato‐associated saprophytes, including the genera Pseudomonas, Bacillus, Enterobacter and Flavobacterium. All bacteria tested produced conductance responses in Special Peptone Yeast Extract, whereas in minimal medium with L‐asparagine only Erwinia spp. and Pseudomonas spp. were able to generate large conductance responses. In minimal medium supplemented with glucose and trimethylamine‐N‐oxide only Enterobacteriaceae, Erwinia spp. included, generated conductance responses, while with pectate as sole carbon source only Erwinia spp. produced distinct conductance responses. The pectate medium proved to be particularly useful for specific automated conductimetric detection of Erwinia spp. in potato peel extracts. Within 48 h, the detection threshold of the conductimetric assay for Eca varied between 102 and 103 cfu per ml peel extract at both incubation temperatures of 20° and 26°C. Ech was detected at concentrations of 104–105 or 103–104 cfu ml‐1 at 20° and 26°C, respectively. To eliminate ‘false’‐positive reactions in conductimetry caused by Erwinia carotovora ssp. carotovora, results of the conductance measurements have to be confirmed by other techniques, like serology or DNA assays.

The development of a conductimetric assay for automated detection of metabolically active soft rot Erwinia spp. in potato tuber peel extracts

The development of a conductimetric assay for automated detection of metabolically active soft rot Erwinia spp. in potato tuber peel extracts

Modified indirect conductimetric technique for detecting low populations of yeasts in beverage concentrates and carbonated beverages

The indirect conductimetric method for detecting yeast activity in foods was modified to increase sensitivity and rapidity for detecting yeast activity in preserved beverage concentrates and carbonated beverages. Nine strains of Zygosaccharomyces and Brettanomyces were isolated from spoiled beverage concentrates and carbonated beverages. A 10 fold dilution of inoculated beverage concentrate in 0·5% tryptone glucose yeast extract broth supplemented with 0·1% Tween 80, followed by pre-incubation under constant aeration at 25°C for 24 h before subjecting to conductimetric assay, significantly ( P −1 –10 o cfu ml −1 in beverage concentrates can be detected within 72–144 h. The threshold limit for detection is in the range of 10 4 –10 5 cells ml −1 . Low populations (one per 10 ml) of yeasts in carbonated beverages can be detected by collecting cells by filtration before subjecting to conductimetric assay, thus facilitating application of the technique for analysing large volumes of product.

Conductimetric assay of pyroglutamyl peptidase activity

A conductimetric method was applied to assay Streptococcus pyogenes pyroglutamyl peptidase (E.C. 3.4.8.11) activity using pyroglutamylalanine or pyroglutamyltyrosine. A spectrophotometric comparison using pyroglutamyl-β-naphthylamide indicated that the sensitivity was much increased when using conductimetry. The use of a C-protected dipeptide such as pyroglutamylalanineamide also increased the sensitivity. Kinetic parameters have been measured for both dipeptides, with K M values of 0.34 mM and 0.47 mM, respectively, for the alanine and tyrosine derivatives.

Conductimetric Assays for the Hydrolase and Transferase Activities of Phospholipase D Enzymes

Measurement of solution electrical conductance (conductimetry) is a simple direct assay method for the protogenic, hydrolytic reactions catalyzed by all phospholipase enzymes. The technique is especially suitable for assay of phospholipase D (PLD) enzymes where cleavage of zwitterionic substrates reinforces the pH dependent conductance change and allows the method to be used over a much wider pH range than the equivalent titrimetric assay. The ability to detect zwitterion cleavage enables the method to assay reactions in which phospholipase D transfers neutral, or anionic, alcohol species to the zwitterionic substrates phosphatidyl choline and phosphatidyl ethanolamine. The method can follow the sequential attack by different phospholipases and provides a simple technique for investigating the effect of substrate structure on susceptibility to various phospholipase enzymes. The results confirm that PLD from Streptomyces chromofuscus can attack lysophospholipids, but cannot transfer primary alcohols to the phosphatidyl residue, while the PLD from savoy cabbage is an efficient transferase, but cannot attack lysophospholipids. The data suggest that the bacterial PLD fails to act as a transferase because it hydrolyzes the transphosphatidylation products. Some phosphatidyl alcohols are more highly susceptible to PLA2 attack than the parent phosphatidyl choline derivatives.

Serological and conductimetric assays for the detection of Pseudomonas syringae pathovar pisi in pea seeds

Test protocols for detecting Pseudomonas syringae pv. pisi, the causal agent of bacterial blight, in pea seeds are generally based on dilution‐plating assays. These assays are usually very specific and reliable, but are time‐consuming and laborious. Tests suited for large scale screening of seed lots are therefore needed. Conductimetric assays, immunofluorescence microscopy (IF) and an enzyme‐linked immunosorbent assay (ELISA) for detecting Ps. syr. pv. pisi in pea seed extracts were compared with dilution‐plating by two extraction methods, viz. 6 h soaking of seeds and 2 h soaking of flour of ground pea seeds in water. In general, the detection of Ps. syr. pv. pisi with conductimetric, IF and dilution‐plating assays in the suspension water of the ground and 2 h‐soaked pea samples was less sensitive than detection in suspension water of the 6 h‐soaked pea seeds. The detection threshold of these assays varied per seed lot between 0 and 4.08 log cfu ml‐1 for the 6 h soaking procedure. The detection threshold of ELISA varied for both extraction methods generally between 4.08 and 6.08 log cfu ml‐1. Detection times recorded in conductimetric assays correlated well (— 0.89 < r < —0.98) with the log colony‐forming units of Ps. syr. pv. pisi added to seed extracts at 27 as well as 17°. However, confirmation of results by isolation on semi‐selective media after conductimetry was more successful at 17° than at 27°, because of the relatively lower activity of saprophytic Pseudomonas spp. at this temperature.

Conductimetric assay of elastase in the supernatants of cultures of Pseudomonas aeruginosa strains

A conductimetric assay of Pseudomonas aeruginosa elastase activity was developed, using either a synthetic peptide (tetraalanine) or unmodified insoluble elastin as substrate. Using purified elastase for calibrations, the activities of 45 culture supernatants of strains from patients with cystic fibrosis were determined. A linear relationship was obtained between the two assays, with a slope of 0.98. These results clearly indicate that measurement of the production of elastase is possible by using a conductimetric method. Whereas elastin represents a more selective substrate (not cleavable by P. aeruginosa alkaline proteinase), tetraalanine is a very convenient substrate if routine and/or fast assays are required.

An investigation of indirect conductimetry for detection of some food‐borne bacteria

Indirect conductimetry using a rapid automated bacterial impedance technique was investigated. Strains of Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and Salmonella spp. grown in Whitley Impedance broth all elicited indirect conductimetric changes. These indirect conductance responses were improved by the addition of 2 g/l glucose to the medium and resulted in maximum changes of 2340-4300 microS with associated maximum rates of change of 520-1210 microS/h. Furthermore, the indirect conductimetric assay detected growth of staphylococci, listeria and salmonella in media containing high concentrations of salts used as selective agents in culture media for the isolation of these organisms.

Formulation of Culture Media for Conductimetric Assays: Theoretical Considerations

Summary: The basic theory of electrolytic conductivity in solutions is described and a model is proposed which allows the direction and relative rates of change of conductivity in microbial cultures to be predicted. Guidelines are presented to enable nutrients to be selected so as to maximize conductivity changes. It is shown that a major consideration in any strategy to maximize conductivity changes in cultures must be to direct as many metabolic activities as possible to act in concert in the production or consumption of protons, and to combine this with use of a pH buffer that exhibits a large change in conductivity on taking up or losing a proton. The ability to predict conductivity changes in microbial systems should permit the rational design of culture media for the selective enumeration of microbes by conductimetric methods and the development of other kinds of conductimetric assays.