A methodology for rapid detection of Salmonella typhimurium using label-free electrochemical impedance spectroscopy

A pathogen detection methodology based on Bayesian decision theory has been developed for rapid and reliable detection of Salmonella typhimurium. The methodology exploits principles from statistical signal processing along with impedance spectroscopy in order to analytically determine the existence of pathogens in the target solution. The proposed technique is validated using a cost-effective and portable immunosensor. This device uses label-free, electrochemical impedance spectroscopy for pathogen detection and has been demonstrated to reliably detect pre-infectious levels of pathogen in sample solutions. The detection process does not entail any pathogen enrichment procedures. The results using the proposed technique indicate a detection time of approximately 6min (5min for data acquisition, 1min for analysis) for pathogen concentrations in the order of 500CFU/ml. The detection methodology presented here has demonstrated high accuracy and can be generalized for the detection of other pathogens with healthcare, food, and environmental implications. Furthermore, the technique has a low computational complexity and uses a minimal data-set (only 30 data-samples) for data analysis. Hence, it is ideal for use in hand-held pathogen detectors.     

Variations in plasmid content during Escherichia coli cultivations detected by on-line flow injection processing

An integrated flow injection process for analysis of intracellular components of microbes has been used to monitor plasmid content in Escherichia coli cultivations inoculated with cells subcultured in the presence or absence of ampicillin. The system allows sampling, sample handling, cell disruption, separation of intracellular components, and analysis in a semi-on-line mode of operation. The time scale for the assay is in the range 15 min (plasmid peak) to 25 min (complete assay cycle). As expected, lower initial plasmid content was found using an inoculum subcultured in the absence of ampicillin. More importantly, significant decrease in plasmid content was detected in the later stages of the cultivations (grown in ampicillin containing medium) even when using inoculum subcultured in the presence of ampicillin. This illustrates the versatility of the system, which allows monitoring of plasmid content as the cultivation proceeds.     

Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus

A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase. Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations. Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye. The dissociation constants of the enzyme-dye complexes were 7.2 +/- 0.2 microM and 11.2 +/- 0.2 microM at 5 degrees C and 20 degrees C respectively.     

A microbial biosensor based on Yarrowia lipolytica for the off-line determination of middle-chain alkanes

A microbial biosensor based on immobilised psychrotrophic yeast Yarrowia lipolytica integrated to FIA for the determination of middle chain alkanes was developed. The system responded very well to middle chain alkanes even at low operational temperatures down to +5 degrees C. The maximum sensitivity was obtained at 15 degrees C. A linear relationship was observed between the sensor response and dodecane concentration up to 100 microM.     

Laser impact assessment in a biofilm-forming bacterium Pseudoalteromonas carrageenovora using a flow cytometric system

Impact by pulsed laser irradiations from an Nd:YAG laser on the marine biofilm-forming bacterium Pseudoalteromonas carrageenovora has been studied using a flow cytometric system. The biofilm-forming bacteria in the planktonic state have been irradiated while flowing, and the mortality and bacterial attachment have been determined by exposing TiN coupons in the system. Coupons suspended in the non-irradiated bacterial flow were treated as the control. The fluence used in the study was 0.1 J/cm(2). Three flow rates (14, 28, and 42 cm/min) and two exposure durations (15 and 30 min) were tested. The results showed the increase in bacterial mortality with the decrease in flow rate. The maximum mortality of 27.5% was observed when the flow rate was 14 cm/min. The bacterial attachment increased with the increase in flow rate and exposure duration. The area of bacterial attachment on the experimental coupons exposed to the irradiated sample was significantly lesser than that for the nonirradiated sample. The results thus show in a flowing system, low power pulsed laser irradiations could reduce the bacterial attachment even though it did not cause significant mortality.     

Induction of phenylpropanoid metabolism by Pseudomonas fluorescens against tomato spotted wilt virus in tomato

Pseudomonas fluorescens (two native strains, one collection strain and their strain mixtures in all possible combinations) when applied through seed, seedling dip, soil and on leaf significantly reduced the tomato spotted wilt virus (TSWV) disease. InP. fluorescens-treated plants, the peroxidase and phenylalamine ammonia-lyase activity increased. Accumulation of phenolic compounds and lignin were shown to be increased in theP. fluorescens-treated plants. Isoperoxidase native PAGE indicated that the peroxidase isoforms in tomato plants induced by fluorescent pseudomonads were different from the control plants; this suggests that the general phenylpropanoid pathway is probably stimulated in tomato plants treated which in turn led to significant reduction in TSWV.     

Laser impact on marine planktonic diatoms: an experimental study using a flow cytometry system

A flow cytometry system was used to evaluate the impact of pulsed laser irradiations from an Nd:YAG laser on two marine coastal water diatoms, Chaetoceros gracilis and Skeletonema costatum. Three flow speeds, i.e. 9, 18 and 27 ml min-1 and three laser fluences, i.e. 0.025, 0.05 and 0.1 J cm-2 pulse-1 were tested during this study. The reduction in cell density and chlorophyll a (chl a) concentrations were monitored by reference to non-irradiated samples as controls. Upon irradiation, the cell density and the chl a concentrations became reduced significantly compared to the control (one way ANOVA p < 0.001 for the cell density in both the species and p < 0.05 for chl a concentrations in both species). A maximum mortality of 0.77 log10 (about 83%) for C. gracilis and 0.68 log10 (about 78%) for S. costatum was observed at 9 ml min-1 flow speed and 0.1 J cm-2 laser fluence. The maximum reduction observed in the chl a concentration was about 26% (control 0.413 and sample 0.306 mg ml-1) for C. gracilis and 27% (control 0.222 and sample 0.16 mg ml-1) for S. costatum, when the flow rate was 9 ml min-1 and the fluence 0.1 J cm-2. In general, mortality increased with an increase in the laser fluence. The results thus show if the cooling water is laser-irradiated to mitigate biofouling, this could result in significant damage to the planktonic flora of the flowing seawater system, which in turn might reduce algal biofilm formation on industrially important structures. The reduction in the chl a concentration showed that the laser irradiations also could result in a significant reduction in the primary productivity of the cooling water.     

Lethal and sub-lethal impacts of pulsed laser irradiations on the larvae of the fouling barnacle Balanus amphitrite

Laboratory experiments were conducted to study the impact of laser irradiation on the larvae of the fouling barnacle Balanus amphitrite. Research pertaining to fouling invertebrate larvae-laser interaction is sparse and, hence, data on this aspect were thought significant in order to consider pulsed low power laser irradiations as a possible future antifouling tool. Lethal and sub-lethal impacts of four very low laser fluences, viz. 0.013, 0.025, 0.05 and 0.1 J cm-2 for three different durations, viz. 2, 10 and 30 s were investigated. Three growth stages of barnacle larvae, viz. nauplii stage II, nauplii stage IV and cyprids were exposed to the mentioned laser fluences for different durations. While lethal impact was assessed immediately after and 1 d after irradiation, sub-lethal impacts were studied by monitoring the success rate of the irradiated nauplii in reaching the cyprid stage. In addition, the swimming speed of VIth stage nauplii after irradiation was studied. In the case of cyprids, in addition to the mortality measurement immediately after and 1 d after irradiation, the settlement rate was investigated. In all the above experiments, non-irradiated larvae served as controls. The results showed an increase in mortality with increasing laser fluence and duration of irradiation. Irradiation for 2 s resulted in significant mortality in nauplii, while it was less in the case of cyprids. In IInd stage nauplii, the mortality immediately after irradiation for 2 s varied from 14.8 +/- 2.12 to 97.1 +/- 4.1% for laser fluences of 0.013 and 0.1 J cm-2, respectively. However, in cyprids, the mortality immediately after irradiation for 2 s varied from 12.2 +/- 3 to 13.4 +/- 1.2% for fluences of 0.013 and 0.1 J cm-2, respectively. The mortality in IVth stage nauplii was less than that for IInd stage nauplii but more than that for cyprids. There was a significant increase in mortality with time after irradiation. The formation of cyprids from the irradiated larvae was significantly less than that observed for non-irradiated larvae. Also, the irradiated larvae showed a significantly slower swimming speed compared to the control samples. The settlement rate in cyprids was reduced significantly by the laser irradiation. This was true even for the lowest fluence and shortest period of irradiation tested. Thus, the results of the experiment showed that even a low power pulsed laser irradiation of 0.013 J cm-2 for 2 s can cause significant damage to fouling barnacle larvae.     

Genetic control of tolerance to type II collagen and development of arthritis in an autologous collagen-induced arthritis model

T cell recognition of the type II collagen (CII) 260-270 peptide is a bottleneck for the development of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. We have earlier made C3H.Q mice expressing CII with glutamic acid instead of aspartic acid at position 266 (the MMC-C3H.Q mouse), similar to the rat and human CII epitope, which increases binding to MHC class II and leads to effective presentation of the peptide in vivo. These mice show T cell tolerance to CII, but also develop severe arthritis. The present investigation shows that non-MHC genes play a decisive role in determining tolerance and arthritis susceptibility. We bred MMC into B10.Q mice, which display similar susceptibility to CIA induced with rat CII as the C3H.Q mice. In contrast to MMC-C3H.Q mice, MMC-B10.Q mice were completely resistant to arthritis. Nontransgenic (B10.Q x C3H.Q)F(1) mice were more susceptible to CIA than either of the parental strains, but introduction of the MMC transgene leads to CIA resistance, showing that the protection is dominantly inherited from B10.Q. In an attempt to break the B10-mediated CIA protection in MMC-transgenic mice, we introduced a transgenic, CII-specific, TCR beta-chain specific for the CII(260-270) glycopeptide, in the highly CIA-susceptible (B10.Q x DBA/1)F(1) mice. The magnification of the autoreactive CII-specific T cell repertoire led to increased CIA susceptibility, but the disease was less severe than in mice lacking the MMC transgene. This finding is important for understanding CIA and perhaps also rheumatoid arthritis, as in both diseases MHC class II-restricted T cell recognition of the glycosylated CII peptide occurs.     

Superporous agarose monoliths as mini-reactors in flow injection systems

A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15×5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 m in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 Å) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterised with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 ml min^–1). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular -galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against -galactosidase).