Multichannel Pipettes Research Articles

Anti-Listeria efficacy of peroxyacetic acid in bacterial buffered solution and on bell peppers

This study aims to 1) evaluate the efficacy of peroxyacetic acid (PAA) to inactivate Listeria monocytogenes in 0.1 % buffered-peptone-water; and 2) evaluate the reduction and mitigation of cross-contamination on peppers. In study-1, aliquots of 1.0-ml PAA solutions (133, 265, and 448 ppm) were added to the first 6 wells of 8-strip-deep well microplates. Then, 0.1 ml of serially diluted nalidixic-acid-resistant (NaL) L. monocytogenes was added and mixed immediately with a multichannel pipette. After exposure for 0, 5, 15, 30, and up to 120-s, 1 ml of 2 × D/E neutralized solution was added to terminate the reaction followed by spread-plating onto tryptic soy agar +200 ppm NaL and incubated at 35 °C for 48 h. In study-2, 5 inoculated red peppers were triple-washed with 15 uninoculated green peppers with 0, 23, 53, and 96 ppm of PAA and analyzed using MPN-method. L. monocytogenes counts were <0.3 log10 CFU/ml after exposure to 133, 265, and 448 ppm of PAA for 120, 90 and 60 s, respectively and fit the Linear and Weibull Models. Applying 23–96 ppm of PAA reduced L. monocytogenes by 2.03–3.32 log10MPN/g and transferred cross-contaminated cell counts by 2.36–3.17 log10MPN/g, with no differences (P > 0.05) compared to the water only treatment. Results suggested that applying allowable concentrations (23–96 ppm) of PAA during triple-wash does not show promising anti-Listeria activity on peppers. Future studies are needed to determine the minimum concentrations of the PAA solution required to effectively reduce and mitigate microbial cross-contamination of Listeria monocytogenes on peppers.

Automated 384-well SYBR Green Expression Array for Optimization of Human Induced Pluripotent Stem Cell Differentiation.

Cell populations and tissues exhibit unique gene expression profiles, which allow for characterizing and distinguishing cellular subtypes. Monitoring gene expression of cell type-specific markers can indicate cell status such as proliferation, stress, quiescence, or maturation. Quantitative reverse transcriptase PCR (qRT-PCR) allows quantifying RNA expression of cell type-specific markers and distinguishing one cell type from another. However, qRT-PCR methods such as TaqMan technology require fluorescent reporters to characterize target genes and are challenging to scale up as they need different probes for each reaction. Bulk or single-cell RNA transcriptomics is time-consuming and expensive. Processing RNA sequencing data can take several weeks, which is not optimal for quality control and monitoring gene expression, e.g., during a differentiation paradigm of induced pluripotent stem cells (iPSCs) into a specialized cell type. A more cost-effective assay is based on SYBR Green technology. SYBR Green is a nucleic acid dye that binds to double-stranded DNA, absorbs blue light at 497 nm, and emits green light at 520 nm up to 1,000-fold upon intercalation with double-stranded DNA. Amplification of a region of interest can be quantified based on the level of fluorescence intensity when normalized to a housekeeping gene and compared to control conditions. Previously, we established a SYBR Green qRT-PCR protocol to characterize samples using a limited set of markers plated on a 96-well plate. Here, we optimize the process and increase throughput to a 384-well format and compare mRNA expression to distinguish iPSC-derived neuronal subtypes from each other by increasing the number of genes, cell types, and differentiation time points. In this protocol, we develop the following: i) using the command-line version of Primer3 software, we design primers more easily and quickly for the gene of interest; ii) using a 384-well plate format, electronic multichannel pipettes, and pipetting robots, we analyze four times more genes on a single plate while using the same volume of reagents as in a 96-well plate. The advantages of this protocol are the increased throughput of this SYBR Green assay while limiting pipetting errors/inconsistencies, reagent use, cost, and time. Graphical overview.

Streptavidin-Coated Solid-Phase Extraction (SPE) Tips for Antibody Phage Display Biopanning.

Phage display is a technique that allows the presentation of unique proteins on the surface of bacteriophages. The phage particles are usually screened via repetitive rounds of antigen-guided selection and phage amplification. The main advantage of this approach lies in the physical linkage between phenotype and genotype. This feature allows the isolation of single unique clones from a panning campaign consisting of a highly diverse population of clones. Due to the high-throughput nature of this technique, different approaches have been developed to assist phage display selections. One of which involves utilizing a streptavidin-coated solid-phase extraction (SPE) tip that is mounted to an electronically controlled motorized multichannel pipette. In this chapter, we will entail the procedures involved in the adaptation of a commercial SPE tip (MSIA™ streptavidin D.A.R.T's®) as the solid phase. This protocol is an updated version of a previous protocol with some minor refinements.

Interfacing centrifugal microfluidics with linear-oriented 8-tube strips and multichannel pipettes for increased throughput of digital assays.

We present a centrifugal microfluidic cartridge for the eight-fold parallel generation of monodisperse water-in-oil droplets using standard laboratory equipment. The key element is interfacing centrifugal microfluidics with its design based on polar coordinates to the linear structures of standard high-throughput laboratory automation. Centrifugal step emulsification is used to simultaneously generate droplets from eight samples directly into standard 200 μl PCR 8-tube strips. To ensure minimal manual liquid handling, the design of the inlets allows the user to load the samples and the oil via a standard multichannel pipette. Simulation-based design of the cartridge ensures that the performance is consistent in each droplet generation unit despite the varying radial positions that originate from the interface to the linear oriented PCR 8-tube strip and from the integration of linear oriented inlet holes for the multichannel pipettes. Within 10 minutes, sample volumes of 50 μl per droplet generation unit are emulsified at a fixed rotation speed of 960 rpm into 1.47 × 105 monodisperse droplets with a mean diameter of 86 μm. The overall coefficient of variation (CV) of the droplet diameter was below 4%. Feasibility is demonstrated by an exemplary digital droplet polymerase chain reaction (ddPCR) assay which showed high linearity (R2 ≥ 0.999) across all of the eight tubes of the strip.

A Simple High-Throughput Field Sample Preparation Method Based on Matrix-Induced Sugaring-Out for the Simultaneous Determination of 5-Hydroxymethylfurfural and Phenolic Compounds in Honey.

In the present work, a high-throughput field sample preparation method was reported for the simultaneous determination of 5-hydroxymethylfurfural and phenolic compounds in honey. Combining a simple and green homogenous liquid−liquid extraction, matrix-induced sugaring-out, with the use of a 96-deepwell plate and multichannel pipette, the proposed method showed its merits in instrument-free and high-throughput preparation. Due to the high-throughput property, the parameters of the method were rapidly and systematically studied using a constructed 4 × 2 × 4 × 3 array (sample amount × ratio of ACN:H2O × standing time × replicates) in a 96-deepwell plate. Analytical performance was fully validated, and the limits of detection and limits of quantification were in the range of 0.17−1.35 μg/g and 0.51−4.14 μg/g, respectively. Recoveries were between 83.98 and 117.11%, and all the precisions were <5%. Furthermore, the developed method was successfully applied in the outdoor preparation of commercial honey samples and the in-field preparation of raw honey samples in apiary. The current work presented a simple, rapid, and high-throughput method for the field sample preparation of honey and provides a valuable strategy for the design of field and on-site sample preparation.

High-throughput salting-out assisted liquid-liquid extraction using a 3D printed device and its application in the quantification of ibrutinib and its metabolite PCI-45227 in human serum

Liquid–liquid extraction methods are widely used for sample treatment in bioanalysis, although their implementation poses a common speed-limiting step in the analytical process when fast separation and detection methods such as UHPLC-MS are used. This study aimed to develop high-throughput salting-out assisted liquid–liquid extraction on a 96-well plate in combination with fast LC-MS analysis of ibrutinib and its active metabolite PCI-45227 (dihydrodiol ibrutinib) in human serum. A specially designed 3D printed extraction device developed in our laboratory allowed for the precise and rapid collection of the organic phase from the 96-well plate using a multichannel pipette, without the risk of aspiration of the bottom aqueous layer. The application of this device significantly accelerated sample preparation and allowed the processing of up to 96 samples in 1 h. The method was successfully validated according to EMA guidelines in the concentration range of 0.1–200 ng/mL for both analytes, providing lower limit of quantification at 100 pg/mL. The intra-day accuracy for IBT was in the range of − 1.67–5.67 %, while the inter-day accuracy was in the range of 0.20–6.90 %. The intra-day precision for IBT was in the range of 3.20–4.37 % and 3.13–4.73 % for the inter-day measurement. PCI-45227 showed intra-day accuracy in the range of − 11.2–3.71 % and the inter-day accuracy in the range of − 5.76–0.92 %. The intra-day precision for PCI was in the range of 3.49–7.64 % and 3.63–8.61 % for the measurement between days.In addition to increasing the speed of sample preparation, this method also offers low consumption of the sample and extraction solvent and can be utilized for other similar small-volume in-well plate extractions where organic solvents of lower density than water are used. The method was successfully applied to the analysis of serum samples (n = 5) of patients with chronic lymphoblastic leukaemia at the trough level (ibrutinib concentration range: 1.63–3.78 ng/mL, PCI-45227 concentration range: 1.84–14.02 ng/mL) and 2 h postdose (ibrutinib concentration range: 7.34–89.0 ng/mL, PCI-45227 concentration range: 5.64–124 ng/mL).

A high-throughput magnetic-based pipette tip microextraction as an alternative to conventional pipette tip strategies: Determination of testosterone in human saliva as a proof-of-concept

In this work, a lab-made and affordable high-throughput pipette tip microextraction strategy is presented. In this approach, a magnetic sorbent is quickly dispersed in the sample by means of a disperser solvent and the resulting dispersion is immediately aspirated into a pipette tip containing a small neodymium magnet inside, which entraps the magnetic sorbent containing the analytes. The sample is then discarded, and the subsequent cleaning and desorption steps are conducted by aspirating/dispensing the suitable solvents. This methodology provides satisfactory results when compared with previous pipette tip extraction strategies (i.e., pipette-tip solid-phase extraction and dispersive pipette extraction). Furthermore, it requires a minimum number of aspirating/dispensing cycles, thus benefiting the ergonomics for the operator, and it is not commercially dependent. This new approach has been tested by measuring testosterone levels in saliva of volunteers as a proof-of-concept. In this regard, a magnetic composite made of CoFe2O4 magnetic nanoparticles embedded in octadecyl bonded silica microparticles was employed as sorbent material. Testosterone was quantified by liquid chromatography coupled to tandem mass spectrometry. Under the optimized conditions, low limits of detection and quantification (7.5 ng L−1 and 24.8 ng L−1, respectively), good repeatability (RSD <9%) and relative recoveries between 82 and 106% were obtained with just one adsorption and two desorption cycles, which requires few seconds per sample. In order to increase even more the sample throughput, a multichannel pipette was also evaluated.

Rapid Method to Quantify the Antiviral Potential of Porous and Nonporous Material Using the Enveloped Bacteriophage Phi6.

The pandemic revealed significant gaps in our understanding of the antiviral potential of porous textiles used for personal protective equipment and nonporous touch surfaces. What is the fate of a microbe when it encounters an abiotic surface? How can we change the microenvironment of materials to improve antimicrobial properties? Filling these gaps requires increasing data generation throughput. A method to accomplish this leverages the use of the enveloped bacteriophage ϕ6, an adjustable spacing multichannel pipette, and the statistical design opportunities inherent in the ordered array of the 24-well culture plate format, resulting in a semi-automated small drop assay. For 100 mm2 nonporous coupons of Cu and Zn, the reduction in ϕ6 infectivity fits first-order kinetics, resulting in half-lives (T50) of 4.2 ± 0.1 and 29.4 ± 1.6 min, respectively. In contrast, exposure to stainless steel has no significant effect on infectivity. For porous textiles, differences associated with composition, color, and surface treatment of samples are detected within 5 min of exposure. Half-lives for differently dyed Zn-containing fabrics from commercially available masks ranged from 2.1 ± 0.05 to 9.4 ± 0.2 min. A path toward full automation and the application of machine learning techniques to guide combinatorial material engineering is presented.

A high-throughput and cost-effective microplate reader method for measuring persulfates (peroxydisulfate and peroxymonosulfate).

Frequent use of persulfates as oxidants, for in situ chemical oxidation and advanced oxidation processes, warrants the need for developing a fast and efficient method for measuring persulfate concentrations in aqueous samples in the lab and on site. Here, we propose a modified method, based on Liang et al.'s (2008) spectrophotometric method, for measuring both peroxydisulfate (PDS) and peroxymonosulfate (PMS) in the aqueous samples. Our method involves a deep 96-well plate, multi-channel pipettes, a small orbital shaker, and a microplate reader; allowing the preparation and analysis of up to 96 samples in one run. Our proposed method shortens the time by 10 folds, consumes only ∼2% of the original reagents, and generates only ∼2% of the liquid waste compared to the Liang et al.'s method, thus, making our method high-throughput, time-efficient, and cost-effective with reduced environmental impact. The presented microplate reader method is validated in terms of linearity, LOD, LOQ, accuracy, precision, robustness, and selectivity. All the parameters satisfied the acceptance criteria, according to ICH guidelines. The linearity of calibration curves was evaluated by performing the F-test. In general, our method has linear ranges from 20 to 42,000 and 5 to 40,960μM for PDS and PMS, respectively. Accuracy (% recovery) results suggested that the LOD and LOQ based on the standard deviation of y-intercepts of the regression lines were the most reliable. The LOD/LOQ values for PDS and PMS were 14.7/44.1 and 4.6/14.4μM, respectively. The proposed method was also modified to work with a standard cuvette spectrophotometer and was validated. A comparison with the UHPLC analysis of PDS showed that our microplate reader method performed equivalently or even outperformed the UHPLC method, in the presence of common groundwater constituents and organic contaminants.

Open Source 3D Printed ISO 8655 Compliant Multichannel Pipette

Multichannel pipettes are used widely to accelerate research and testing in life sciences laboratories and within the biomedical industry. Commercial multichannel pipettes cost several hundred to thousand U.S. dollars and are not accessible in many areas. This study utilizes an open source and digital distributed manufacturing model to design a USD$24 4-channel 200 micro-liter pipette. The design uses widely-available interchangeable off-the-shelf parts and custom components, which can be fabricated with a low-cost fused-filament RepRap-class desktop 3D printer. The manuscript describes the design, construction, and validation of an ergonomic open-source multichannel pipette, as well as the evaluation of pipetting parameters and quality control. The open source multichannel pipette is found to be compliant with ISO 8655 standards for precision and accuracy. The device has separate syringes that operate individual tips, which prevents cross contamination of specimens because there is no direct contact of different liquids with each other and no mixing with actuating air. Each syringe is easily replaced for different experiments and the source CAD files are provided to enable others to build variants or custom configurations of the open source multichannel pipette. <strong>Metadata Overview</strong> Main design files: <a href="https://osf.io/9tn6e/" target="_blank"><em>https://osf.io/9tn6e/</em></a> Target group: chemistry, biochemistry, biology, biomedical, medical, and associated disciplines Skills required: desktop 3D printing – easy, mechanical assembly – easy Replication: <em><a href="https://www.appropedia.org/Open_Source_3-D_Printed_ISO_8655_Compliant_Multichannel_Pipette" target="_blank">https://www.appropedia.org/Open_Source_3-D_Printed_ISO_8655_Compliant_Multichannel_Pipette</a></em> for comments and feedback. <em>See section “Build Details” for more detail</em>.

Cryoprotectant-free preservation of bacteria using semi-spherical drops

Cryopreservation is a widely used long-term preservation method to ensure the quality and vitality of microbes in laboratories and biological resource facilities. However, freeze-thaw damage and osmotic pressure changes during cryopreservation adversely impacts microbial survival. Significant expenditure of resources and expertise are required to select the right cryoprotectant and optimize its concentration for maximum survival of diverse microorganisms. This work describes a cryopreservation method that obviates the need for cryoprotectants by exploiting the unique thermal characteristics of semi-spherical drops. Here, a plurality of these drops, each 10 μl in volume, created on a highly non-wetting flat-sheet substrate with holes and frozen at −70 °C. Deriving an f (x) metric as a measure of relative viability, storage in drops in the absence of cryoprotectants was found to improve the survivability of Staphylococcus epidermidis by 1.91 times compared with the same sample stored in larger 50-μl volumes in standard 1.5-ml tubes. This also compares well with a value of 2.33 obtained with standard preservation with cryoprotectant. The drop method allows high throughput aliquoting of the bacterial culture into multiple discrete drops using multichannel pipettes or automated liquid handlers and the edges of the holes provides a pinning action that holds the drop stably against gravitational roll-offs. It also allows samples to be removed in discrete small volumes, thus, reducing the number of freeze thaw cycles and associated cell damage. The flat-sheet architecture of the substrate reduces the amount of plastic waste generated and augments green laboratory practices.

Processing Hundreds of SARS-CoV-2 Samples with an In-House PCR-Based Method without Robotics

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System.

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

Counting bacteria in microfluidic devices: Smartphone compatible ‘dip-and-test’ viable cell quantitation using resazurin amplified detection in microliter capillary arrays

Viable bacterial cell counting is fundamental to analytical microbiology and agar plate colony counting remains common yet laborious and slow. Here, we demonstrate two methods for counting bacteria using commercially available microfluidic devices. We show that accurate viable cell counting is possible using simple and easy ‘dip and test’ arrays of microcapillaries. Colorimetric and fluorescent growth detection both permit viable cell counting in microcapillaries either by limiting dilution into multiple microfluidic compartments using a single endpoint measurement, or alternatively by quantifying growth kinetics. The microcapillary devices are compatible with conventional 96 well plates and multichannel pipettes, expanding each microplate row into 120 individual 1 or 2 μL samples. At limiting dilution, counting the proportion of positive compartments permitted accurate calculation of gram-negative and gram-positive bacteria (E. coli and S. saprophyticus) at concentrations down to as low as 10 CFU/mL with almost 1:1 agreement with agar plate colony counts over four orders of magnitude. A smartphone camera was sufficient to record endpoint images of resazurin growth detection both colorimetrically and fluorescently. Viable cell counting of E. coli and S. saprophyticus was also possible through recording growth kinetics and determining the time taken to detect resazurin conversion. However, only the limiting dilution method remained consistent in the presence of urine matrix, as some interference in growth rate was observed when bacteria were spiked into higher concentrations of normal urine to simulate urinary tract infection patient samples. However, with the limiting dilution counting method endpoint growth was always detected even in the presence of 90% urine matrix, suggesting that this method might permit bacterial pathogen counting directly in clinical samples without agar plating.

Sample Preparation for High-Throughput Urine Proteomics Using 96-Well Polyvinylidene Fluoride (PVDF) Membranes.

Proteomics analysis of urine samples allows for studying the impact of system perturbation. However, meaningful proteomics-based biomarker discovery projects often require the analysis of large patient cohorts with hundreds of samples to describe the biological variability. Thus, robust high-throughput sample processing methods are a prerequisite for clinical proteomics pipelines that minimize experimental bias due to individual sample processing methods. Herein we describe a high-throughput method for parallel 96-well plate-based processing of urine samples for subsequent LC/MS-based proteomic analyses. Protein digestion and subsequent sample processing steps are efficiently performed in 96-well polyvinylidene fluoride (PVDF) membrane plate allowing for the use of vacuum manifolds for rapid liquid transfer, and multichannel pipettes and/or liquid handing robots. In this chapter we make available a detailed step-by-step protocol for our 'MStern blotting' sample processing strategy applied to patient urine samples followed by mass spectrometry-based proteomics analysis. Subsequently, we provide an example application using minimal volume of urine samples (e.g. 150μL) collected from children pre and post thoracotomy to identify the predominant sites of protein catabolism and aid in the design of therapies to ameliorate protein catabolism and breakdown during critical illness. Furthermore, we demonstrate how the systemic state is reflected in the urine as an easily obtainable, stable, and safe biofluid.

New and simplified method for drug combination studies by checkerboard assay

To evaluate the effect of two combined antimicrobial drugs, one method currently in use is the checkerboard assay in a 96-well microplate, which gives a good in vivo estimation of the drug-drug combination effect. Appropriate and consolidated methods are described in numerous scientific publications which are, however, in turn, laborious and time-spending, specifically for the setting of the 96-well microplate preparation. Each drug of every combination must be prepared and dispensed individually in several steps, often limiting its use in terms of consumed materials and working time. In our method, the strengths of the previous consolidated techniques are kept, although the toughness and the execution time are drastically reduced. No special laboratory apparatuses are needed. All the procedures of our method can be referred to the CLSI or EUCAST guideline. The method provides few main steps, which can be summarised in:•Preparation of the microorganism inoculum and three concentrations of antimicrobial drugs.•Easy dispensing of all reagents into the microplates with a multichannel pipette.•Evaluation of the microorganism optical density (OD) by a microplate reader, and calculation of growth percentage for each of the 77 combinations.

High-throughput Method for Detecting Siderophore Production by Rhizosphere Bacteria.

Siderophores, a key substance that microorganisms produce to obtain iron under iron-limited conditions, play an important role in regulating interactions between beneficial bacteria and pathogenic bacteria. A large number of bacteria were isolated from the rhizosphere, and we used the method presented here to assay the siderophore production by these rhizosphere bacteria. This method is a modified version of the universal chrome azurol S (CAS) assay that uses a 96-channel manual pipetting workstation. By combining the liquid CAS assay with the multi-channel pipette workstation, high-throughput and rapid detection of siderophore production can be achieved. In summary, this method can be used to gain a general understanding of siderophore production by rhizosphere bacteria.

A modified gentamicin protection assay for detecting invasive phenotype of Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (SE) is one of the most important zoonotic pathogens that cause enteritis and systemic infection in animals and human. Understanding invasive capacities of SE isolates is of vital importance to elucidate pathogenesis of Salmonella infection. To improve the throughput capacity and repeatability of classical gentamicin protection assay (GPA), a modified PGA was developed by taking high-throughput advantage of 96-well cell plates and multichannel pipettes. In addition, drop plate technique rather than spread plate method was applied in the modified GPA protocol for bacterial enumeration. The modified GPA protocol was evaluated by phenotyping intracellular replication of a high virulent and a low virulent SE isolates, JL228 and LN248, in a phagocytic cell line RAW264.7. The protocol was then applied in invasive phenotype determination of 16 SE strains to non-phagocytes (HT-29) and the intracellular replication of 43 SE strains to phagocytes (RAW264.7). Significant lower intra-group and inter-group coefficient of variations of the modified GPA was observed, implying good repeatability and reproducibility over traditional protocol. Further, replication phenotypes were also correlated with those from direct observation by confocal microscopy. Collectively, the improved GPA protocol had advantages of high throughput capacity, good repeatability and reliability, it was also noticed that the protocol also represented a fast and labor-saving alternative scheme for the invasive phenotype determination of Salmonella Enteritidis, and providing reliable phenotype profiles for Salmonella-host interplay interpretation.

Combining In-Tip Reaction and Infrared Thermal Imaging for Fast and Portable Enthalpimetric Analysis.

In this work, the in-tip thermal infrared enthalpimetry (in-tip TIE) method is proposed for fast enthalpimetric analysis. In this method, the reactions inside the tips of a multichannel pipette were combined with temperature monitoring by an infrared camera. The filter paper was used inside the tips to retain reagents as solutions (wetted paper mode) or as solids (dried paper mode) to perform neutralization, redox, or precipitation reactions. The dried reagents inside the tips were obtained by oven drying a solution retained in the filter paper. The determination of the total acidity of the vinegar, ascorbic acid in vitamin C tablets, and chloride in soy sauces and saline inhalation solutions was performed as examples of the application of the proposed method. The agreement with reference methods ranged from 98 to 107%. The use of reagents dried inside the tip was feasible, leading to a simple aspiration of sample solution within the 12 tips of the pipette to perform a rapid analysis (1 min). Therefore, up to 720 measurements in 1 h were feasible for in-tip TIE over up to 12 measurements for the reference methods. Moreover, miniaturization reduced reagent consumption and residue generation. For example, for in-tip TIE, only 3.6 mL of residues was generated (n = 12) over 60-240 mL in reference methods (n = 3). Contrarily to other TIE methods, no microplates or stirring was required, opening possibilities for field analysis since the multichannel pipette and the infrared camera are both operated with batteries.