Rapid Enumeration Research Articles

A novel method for the rapid enumeration of planktonic salmon lice in a mixed zooplankton assemblage using fluorescence

The relative rarity of the planktonic larval stages of salmon lice in comparison to other animals captured in a zooplankton assemblage is an obstacle to estimating their abundance and distribution. Due to the labour intensiveness of standard plankton sorting approaches, the planktonic stages of salmon lice remain understudied and unmonitored despite their importance to the spread of the parasite between salmon farms and to wild salmonids. Alternative methods of identification have been investigated and in a previous study a fluorescence signal was identified. Using filters to target that signal with fluorescence microscopy (excitation/emission wavelengths of 470/525 nm), the salmon louse has a fluorescence intensity 2.4 times greater than non-target animals, which distinguishes it from the zooplankton assemblage and enables rapid enumeration. Here, we present a novel method for the enumeration of planktonic salmon lice larvae, nauplius and copepodid stages, in a mixed zooplankton sample using fluorescence-aided microscopy. Performance of the method was evaluated with a blind trial which found a median accuracy of 81.8% and a mean sample processing time of 31 min. Compared with previously published findings, the novel method provides satisfactory accuracy and enumeration that is more than 20 times faster than traditional light microscopy approaches. Factors influencing the performance of the method are identified and recommendations are made for targeted sampling and automated enumeration.

OnePetri: Accelerating Common Bacteriophage Petri Dish Assays with Computer Vision.

Introduction: Bacteriophage plaque enumeration is a critical step in a wide array of protocols. The current gold standard for plaque enumeration on Petri dishes is through manual counting. However, this approach is not only time-consuming and prone to human error but also limited to Petri dishes with countable number of plaques resulting in low throughput. Materials and Methods: We present OnePetri, a collection of trained machine learning models and open-source mobile application for the rapid enumeration of bacteriophage plaques on circular Petri dishes. Results: When compared against the current gold standard of manual counting, OnePetri was ∼30 × faster. Compared against other similar tools, OnePetri had lower relative error (∼13%) than Plaque Size Tool (PST) (∼86%) and CFU.AI (∼19%), while also having significantly reduced detection times over PST (1.7 × faster). Conclusions: The OnePetri application is a user-friendly platform that can rapidly enumerate phage plaques on circular Petri dishes with high precision and recall.

The Asymmetric Switch Cost between Subitizing and Estimation in Tactile Modality

Subitizing (with number of items less than or equal to 4) and estimation (with number of items equal to or larger than 5) are two types of strategies for rapid enumeration. Debate remains about the relationship between subitizing and estimation, and evidence is scarce from the tactile modality. The present study has developed a novel processing switching paradigm to assess costs in cognitive resources related to switching between the two processes. Participants were required to enumerate two sequentially presented arrays of tactile pins with the set size of each array either within or out of a predetermined subitizing range (1-3 tactile pins). We found a general task-dependent switch cost within a narrow temporal range- the first numerical processing significantly influenced the second numerical processing with stimuli-onset-asychrony (SOA) between the two events was 100 ms. Specifically, the error rate of the second enumeration increased, while the accuracy and the precision decreased, when the enumeration process switched between the two strategies (subitizing-estimation or estimation-subitizing), relative to no switch (subitizing-subitizing or estimation-estimation). At the same time, an asymmetry was observed on the accuracy and the precision— a switch from estimation to subitizing triggered a robust switch cost effect with larger magnitudes than a switch from subitizing to estimation did. Taken together, our results suggest that the relationship of subitizing and estimation in tactile modality is consistent with the findings in visual modality (Anobile et al., 2012; Burr et al., 2010). Specifically, there is an additional mechanism over and above the unitary estimation, manifests certain flexibility and only operates on small numerosities when attentional resources permit.

Optical methods for bacterial detection and characterization

Rapid identification and enumeration of bacteria are critical, given the surge of antibiotic-resistance, global exchange of food products, and the use of bacteria for bioremediation, pharmaceutical, and food production. In response, a wide range of methods are being developed that can be broadly classified as nucleic acid-based, structure-based, mass spectrometry-based, and optically based. Optical methods have generated interest given the potential for rapid, non-destructive, high-throughput, and amplification-free measurements that require minimal sample preparation. This Perspective reviews optical methods, which are applied to identification, enumeration, and greater understanding of bacteria routinely and more importantly at the cutting edge of research, with the aim of identifying gaps and opportunities for development. We have focused primarily on methods that directly measure bacteria and not their effect on the sample matrix or sensing, which requires a biorecognition element (i.e., label specific to some component of the bacterium). We identify gaps in the existing techniques and avenues for innovation. Finally, we suggest the parameters that should be considered and recorded when reporting the development of existing and new methods for bacterial characterization. This Perspective is intended for physicists interested in developing new optical methods for the study of bacteria and microbiologists in need of an optical technique for bacterial applications.

A Combined Immunofluorescence and Fluorescent Viability Cocktail Staining Procedure for Rapid Microscopic Detection and Enumeration of Live Legionella pneumophila.

This report describes a combined immunofluorescence and fluorescence viability stain applied as one staining solution for rapid detection oflive Legionella pneumophila in mixed bacterial populations. Instead of sequential viability staining with the Invitrogen BacLight LIVE/DEAD staining kit followed by antibody-Alexa Fluor (AF) 647 conjugate staining to identify liveL. pneumophila, a combined single cocktail solution staining protocol was developed to simplify and accelerate the time to detection of viable L. pneumophila serogroup-1 (SG-1) in mixed species populations on a filter membrane. The stain cocktail will aid in accelerating fluorescence microscopic analysis of cooling tower, air conditioner and water fountain or other liquid samples for the presence of L. pneumophila and its viability status. Visibly red stained cells were identified as dead non-L. pneumophila SG-1 cells, while green fluorescing cells represented viable non-L. pneumophila SG-1 cells. Due to also staining red with antibody-AF 647, L. pneumophila SG-1 cells were pseudocolorized as blue to distinguish them from other dead cells. Fluorescence color emission mixing from the viability dyes (SYTO 9 and propidium iodide) with antibody-AF 647 stained L. pneumophila led to other fluorescent colors. For example, green plus pseudocolorized blue AF 647-antibody- labeled cells were identified as live cyan-colored L. pneumophila SG-1 cells. Magenta-colored cells resulted from dead L. pneumophila cells that combined red propidium iodide with blue pseudocolorized AF 647-antibody emissions. Analysis of measured RGB (red, green, blue) color values in microscopic images of mixed bacterial populations suggests the possibility of facile automated discrimination of subpopulations of live and dead L. pneumophila and non-L. pneumophila species by computers in 3-dimensional RGB color space after staining in the combined cocktail which will save time for more rapid microscopic detection of potential sources of Legionnaire's disease.

PIECES FRAMEWORK ON THE IMPLEMENTATION OF RAPID ENUMERATION AND EVALUATION INFORMATION SYSTEM OF 2020 POPULATION CENSUS

In September 2020, Statistics Indonesia (BPS RI) carried out a major activity, namely the 2020 Population Census At the SP2020 data processing stage, Mobile Capture is one of the options for photographing data collection results. During the use of mobile capture, BPS West Java Province encountered several obstacles. West Java Province BPS created an application called the Rapid Enumeration dan Evaluation 2020 Population Census Information System (Sicepat32 SLS) to monitor the attainment of enumeration to the smallest level (local environmental unit). To find out whether Sicepat32 SLS is working and functioning properly, it is necessary to evaluate the performance of the information system. The purpose of this study is to measure the level of user satisfaction with the application of Sicepat32 SLS and to assess whether Sicepat32 SLS can meet the needs of users. In this study, the PIECES Framework analysis model will be used and has produced an assessment score in the Performance domain of 4.07; Information and Data of 4.17; Economy 4.1; Control and Security with a score of 4.03; Efficiency with a score of 4.18 and Service getting a score of 4.18

Potato Cyst Nematode Egg Viability Assessment and Preparasitic Juvenile Screening Using a Large Particle Flow Cytometer and Sorter.

Potato cyst nematode (PCN) cysts consist of heterogenous populations of eggs, juveniles, and eggshells that make manual sorting of individual life stages cumbersome. The number of viable PCN eggs is a major determinant of crop damage. An accurate high-throughput PCN egg viability assay is useful for developing effective management and eradication plans. In this study, we present a method for rapid and precise enumeration and sorting of PCN eggs and juveniles, along with an egg viability assessment by staining eggs with the fluorescent stain, acridine orange, and sorting with the Complex Object Parametric Analyzer and Sorter (COPAS) system, a large particle flow cytometer. Both size sorting and fluorescent sorting capabilities of the COPAS were explored. By using the COPAS, sorting efficiency for eggs and preparasitic second-stage juveniles (J2s) was 97.6 and 97.2%, respectively, with 99% recovery at a flow rate of 15 events/s. Purity of sorted live and dead eggs was 95.5 and 94.1%, respectively. Sorting of J2s by size indicated that 15 to 16.4% of Globodera ellingtonae or G. pallida had an average body length of 436.1 ± 3.4 µm compared with an average size of 512.9 ± 4.4 µm for the majority of the J2 population for both species. A red autofluorescing J2 population was also identified through sorting. Sorting of eggs by flow cytometry did not significantly affect hatching (55.1 ± 1.2 and 53.9 ± 1.6%, respectively, for sorted or nonsorted eggs) or juvenile motility (91.3 ± 1.0 or 90.1 ± 1.1%, respectively), thus confirming that the method does not impair the biological activity of the nematode.

Implementation of ATP and Microbial Indicator Testing for Hygiene Monitoring in a Tofu Production Facility Improves Product Quality and Hygienic Conditions of Food Contact Surfaces: A Case Study.

Rapid ATP testing and microbiological enumeration are two common methods to monitor the effectiveness of cleaning and sanitation in the food industry. In this study, ATP testing and microbiological enumeration were implemented at a tofu production facility with the goal of improving cleaning practices and overall plant hygiene. Results from ATP monitoring were used to target areas of the production environment needing additional cleaning; ATP results were verified by microbiological enumeration of aerobic microorganisms, lactic acid bacteria, and yeasts and molds. Products from the production line were enumerated for the same microorganisms to determine if there was an impact on product quality. After the implementation of ATP monitoring and targeted cleaning, there was a statistically lower proportion of swabs that failed to meet established sanitary requirements for ATP, aerobic microorganisms, and lactic acid bacteria (p < 0.05), but not for yeasts and molds. ATP swabs and microbiological enumeration agreed on site hygiene 75.1% (72.3-77.7%, 95% CI) of the time. Product data indicated that unpasteurized finished products contained a statistically lower microbial load of the three groups of organisms following implementation of the practices (p < 0.05).ImportanceCleaning and sanitation are critical to maintaining safe and high-quality food production. Monitoring these activities is important to ensure proper execution of procedure and to assure compliance with regulatory guidelines. The results from monitoring activities can direct targeted cleaning of areas with higher risk of contamination from foodstuffs and microorganisms. The results of this study show that ATP monitoring and microbiological enumeration are useful tools to verify and improve the efficacy of cleaning and sanitation practices, which can have a positive impact on both plant hygiene and product quality. However, testing regimes and critical parameters will vary based on the product and facility.

One-Dimensional Flow of Bacteria on an Electrode Rail by Dielectrophoresis: Toward Single-Cell-Based Analysis.

Many applications in biotechnology and medicine, among other disciplines, require the rapid enumeration of bacteria, preferably using miniaturized portable devices. Microfluidic technology is expected to solve this miniaturization issue. In the enumeration of bacteria in microfluidic devices, the technique of aligning bacteria in a single line prior to counting is the key to an accurate count at single-bacterium resolution. Here, we describe the numerical and experimental evaluation of a device utilizing a dielectrophoretic force to array bacteria in a single line, allowing their facile numeration. The device comprises a channel to flow bacteria, two counter electrodes, and a capture electrode several microns or less in width for arranging bacteria in a single line. When the capture electrode is narrower than the diameter of a bacterium, the entrapment efficiency of the one-dimensional array is 80% or more within 2 s. Furthermore, since some cell-sorting applications require bacteria to move against the liquid flow, we demonstrated that bacteria can move in a single line in the off-axial direction tilted 30° from the flow direction. Our findings provide the basis for designing miniature, portable devices for evaluating bacteria with single-cell accuracy.

Assessment of Glial Fibrillary Acidic Protein Binding to the Surface of Leukocytes with Dark‐Field Imaging and Computational Analysis

AbstractThe glial fibrillary acidic protein (GFAP) is widely established as a traumatic brain injury (TBI) biomarker and can be used in early diagnosis. As essential primary human immune cells, leukocytes are recruited to injured cerebral sites during TBI response, where they can interact with and potentially bind to TBI biomarkers. To date, no studies have demonstrated ultra‐low GFAP binding enumeration on leukocytes. Herein, a dark‐field imaging technique coupled with computational analysis is introduced to quantify GFAP bound to peripheral blood mononuclear cells (PBMCs). Dark‐field microscopy (DFM) with a custom‐written image acquisition software is developed for rapid 3D PBMC imaging by utilizing specific antiGFAP monoclonal antibody functionalized gold nanoparticles (antiGFAP‐AuNPs) as contrast‐generating probes. Subsequently, the developed algorithm is utilized in processing thousands of acquired images for rapid visualization and enumeration of bound antiGFAP‐AuNP on each leukocyte. The proposed method is demonstrates the specific binding of GFAP to the surface of PBMCs on a healthy donor blood. Thereafter, subpopulations of PBMCs with antiGFAP‐AuNP binding are identified with the assistance of fluorescence imaging and DFM imaging, paving a new way to understanding the relationship between TBI and leukocyte classes. Hence, this study offers a rapid and ultra‐sensitive strategy for biomarker assessment following TBI.

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR.

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 104 spores/bee, and the stable detection level was ≥ 2.40 × 105 spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 104 copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows\u2019 milk

Bacteriophage-based methods for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) in veterinary specimens are a recent addition to the Johne’s disease diagnostic toolbox. Here, we report the use of D29 mycobacteriophage-coated tosylactivated paramagnetic beads to capture and concentrate MAP cells from samples (termed phagomagnetic separation, PhMS) and then naturally lyse viable MAP cells (from the inside out) to provide DNA for IS900 qPCR purposes. Transmission electron microscopy confirmed that D29 phages had bound to beads in the correct orientation and that the phage-coated beads captured MAP cells from a suspension. During test optimization, conventional IS900 PCR results were used to subjectively assess the effect of different phage:bead coating ratios, differing amounts of coated beads during PhMS, optimal incubation time post-PhMS to obtain maximal MAP DNA, and the potential benefit of a brief heat shock (55 °C/1 min) prior to IS900 TaqMan qPCR. The limit of detection 50% (LOD50%) of the optimised PhMS-qPCR assay was 10.00 MAP cells/50 ml milk (95% CI 1.20–82.83). Finally, in order to demonstrate the new assay’s ability to detect viable MAP in naturally contaminated milk, bulk tank milk samples from 100 dairy farms were tested. Forty-nine (49%) of these tested PhMS-qPCR-positive, with viable MAP numbers detected ranging from 3–126 MAP/50 ml. The novel PhMS-qPCR assay is a sensitive, specific and easy-to-apply phage-based assay for viable MAP, with potential application for milk surveillance or diagnosis of Johne’s disease.Key points• Phage-coated magnetic beads could capture, concentrate and lyse MAP cells from milk.• PhMS-qPCR assay proved to be a rapid, sensitive and specific test for viable MAP.• A potential application of PhMS-qPCR assay for milk surveillance was demonstrated.

Using direct epifluorescent microscopic count for rapid enumeration of viable yeast and bacteria in injured conditions

Traditional method for assessing cell count requires an incubation period of 2-3 days. The direct microscopic count (DMC) method gives rapid enumeration of yeast and bacteria. However, the application is limited, since it is not possible to distinguish accurately between viable and nonviable cell. In this report, several nucleic acid dyes from Molecular Probe Inc. were used to stain the cells for enumeration of live and dead yeast in injured conditions, in which the dye can stain the DNA of nucleus and mitochondria, which then fluoresce yellow under fluorescence microscope, as well as stain the cytoplasm of actively growing cells to fluoresce green, while the cytoplasm of inactive cells fluoresced orange. The direct microscopic count multiplied by the live cell count ratio obtained by this direct epifluoroscent microscopic count (DEMC) method gives the estimated viable yeast count. According to our results, the correlation coefficient (R2) between the cell count obtained by the DEMC method and that by the standard plate count was 0.91 for yeast and 0.96 for bacteria in frozen and heating conditions, respectively. Requiring only 30 min, this method can also be used for the rapid enumeration of viable bacteria in injured conditions.

Abstract B30: Clinical correlation of circulating tumor cells as a blood marker in Indian head and neck cancer patients

Abstract Objectives: To establish a rapid, highly specific, efficient, sensitive, and affordable CTC enumeration liquid biopsy technology and to validate its efficacy to isolate CTC disseminating from epithelial tumors of HNC subpopulation in India. Furthermore, to study the correlation of CTC distribution from peripheral blood with respect to various clinicopathologic factors in these patients. Materials and Methods: A cross-sectional study was conducted using peripheral blood from enrolled 350 HNC patients. CTC were isolated using DCGI, India-approved technology, that exploits EpCAM-based immunomagnetic separation. EpCAM+ tumor cells were isolated from only 1.5 mL blood and critically assayed for cytokeratin 18 (CK18) expression and quantified using fluorescence imaging of CTCs so as to obtain a threshold to further minimize nonspecific and false-positive enumeration. CTC enumeration was subsequently subjected to statistical correlation with various clinical and pathologic parameters. Results: We detected CTCs from all HNC patients across its various subsites, and there was a minimum threshold of at least 12 CTC in early oral cancer patients according to their clinicopathologic signatures. Compared to early oral cancer patients, advanced nodal patients showed 40% escalation in CTC count, while up to 80% increase in CTC count was observed when associated with aggressive features such as lymphovascular emboli (LVE) and with extranodal extensions. Of note, laryngopharyngeal primary had the highest mean CTC count of 33 in 1.5mL blood. Conversely, patients with advanced disease had higher CTC count and this was staggered in comparison with nearly but not all of the clinical features. Remarkably, higher clinical N (nodal) stage statistically correlated with increased CTC count and a marked increase in CTC was seen in tumors that showed lymphovascular emboli on histopathology and extranodal extension. The CTC counts were independent of parameters such as the age, sex, T stage, perineural invasion, bone involvement, or skin involvement. There was a notable trend towards reduced CTC count after chemotherapy; however, it was not statistically significant. Conclusion: Our rapid and efficient CTC platform has demonstrated and clinically validated its use in Indian HNC phenotypes. This is the first comprehensive study to show a staggering positive correlation of CTC with various clinicopathologic factors. It comprised the largest number of oral cancer patients throughout the entire spectrum of HNSCC, the most common cancer in India. High CTC counts among HNC patients could possibly be one of the reasons for their dismal outcomes, and further studies with a correlation of CTC with patient survival in HNC are warranted. However, this study strongly implicates a perspective utility of CTC as a tumor marker in establishing the clinical staging in HNC patients. Citation Format: Jayant Khandare, Burhanuddin Nuruddin Qayyumi, Atul Bharde, Gourishankar Aland, Ajit Sagare, Swati Tripathi, Nitin Singh, Sreeja Jayant, Ashish Muglikar, Reecha Badave, Aravindan Vasudevan, Kumar Prabhash, Pankaj Chaturvedi. Clinical correlation of circulating tumor cells as a blood marker in Indian head and neck cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B30.

Effectiveness of Nitrogen Dioxide Fumigation for Microbial Control on Stored Almonds

Quality of stored almonds is compromised by insect infestations and microbial contamination. Nitric oxide (NO) is a potent fumigant for postharvest pest control on fresh and stored products. NO fumigation must be conducted under ultralow oxygen conditions, and it always produces nitrogen dioxide (NO2), depending on the O2 level in the fumigation chamber. NO and NO2 have proven antimicrobial effects but have not been tested for efficacy against microbes in almonds. We evaluated, in this study, fumigation of unpasteurized almonds with NO2 at different levels for inhibition of bacteria and fungi. Almonds were fumigated with 0.1, 0.3, or 1.0% NO under ambient O2 to generate 0.1, 0.3, or 1.0% NO2 conditions; the fumigation treatments lasted 1 or 3 days at 25°C. GreenLight rapid enumeration tests on diluted wash-off almond samples from NO2 fumigation treatments showed either greatly reduced microbial loads or complete control of microorganisms, depending on NO2 concentration and treatment duration. NO2 fumigation was more effective against fungi than against bacteria. These results suggest that postharvest NO fumigation with proper levels of NO and NO2 can be used for insect and microorganism control on stored almonds.

Evaluation of the 3M™ Petrifilm™ Rapid E. coli/Coliform Count Plate for the Enumeration of E. coli and Coliforms: Collaborative Study, First Action: 2018.13.

The 3M™ Petrifilm™ Rapid E. coli/Coliform Count Plate is a selective and differential sample-ready-culture medium designed for the rapid enumeration of Escherichia coli (E. coli) and coliforms in the food and beverage industries. The 3M Petrifilm Rapid E. coli/Coliform Count Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Chapter 4 Enumeration of Escherichia coli and the Coliform Bacteria, the International Organization of Standards (ISO) 4832:2006 Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coliforms-Colony-count technique, and ISO 16649-2:2017 Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli-Part 2 Colony-count technique at 44 degrees C using bromo-4-chloro-3- indolyl beta-D-glucuronide methods for the enumeration of E. coli and coliforms in dry dog kibble. The candidate method was evaluated using two diluents, Butterfield's phosphate buffered diluent and peptone salt solution, in a paired study design with each reference method in a multi-laboratory collaborative study following the current AOAC Validation Guidelines. Three target contamination levels and an uninoculated control level were evaluated. The candidate and reference methods were not statistically different at each contamination level. Reproducibility values obtained during the collaborative study were similar between the candidate and reference methods. These results demonstrate that the candidate method is equivalent to the reference methods. 3M Petrifilm Rapid E. coli/Coliform Count Plate was recommended for Official First Action status for enumeration of E. coli and coliforms in a broad range of foods and environmental surfaces.

Cell Enumeration of Leptospira by Flow Cytometry.

Rapid and reliable enumeration of Leptospira spp., the causative agent of leptospirosis, represents a technical challenge because leptospires are thin, highly motile, and slow-growing bacteria. The current gold standard for cell enumeration is the use of a Petroff-Hausser counting chamber and a dark-field microscope, but this method remains time-consuming and lacks reproducibility. New alternative techniques are then of great interest. Here we describe the protocol for counting leptospires by flow cytometry. This method is rapid, reproducible, sensitive, and hence suitable to become a new standard to enumerate Leptospira spp.

Nitrogen Dioxide Fumigation for Microbial Control on Unshelled Peanuts

Stored peanuts often need treatments to control microbial infections as well as insects to maintain postharvest quality. Nitric oxide (NO) is a recently discovered fumigant for postharvest pest control. NO fumigation must be conducted under ultralow oxygen condition to preserve NO and always contains NO2 due to NO reaction with oxygen and NO2 has antimicrobial property. Therefore, NO fumigation has potential to control both pests and pathogens. In this study, we evaluated antimicrobial effects of NO2 fumigation on unpasteurized unshelled peanuts. Peanuts were fumigated with 0.3%, 1.0%, and 3.0% NO2 for three days at 25˚C by injecting NO gas into glass jars to react with O2 in the atmosphere. After fumigation, wash-off microbial samples were collected from intact peanut samples and, then, cracked open peanut samples with non-selective tryptic soy broth medium. The wash-off samples were then diluted with both the non-selective medium and a fungal-selective potato dextrose broth medium and were tested on GreenLight™ rapid enumeration test based on oxygen depletion on culture medium. All three NO2 fumigation treatments showed significant antibacterial and antifungal effects on intact peanuts as well as on cracked peanuts with complete inhibition with 3.0% NO2. Fumigation did not have obvious effects on appearance of skinned peanut kernels. These results suggested that NO2 fumigation has potential to control microbes on stored products, and NO fumigation with the combination of NO and NO2 has potential to control both insects and microbes on stored products.

A disposable gold-cellulose nanofibril platform for SERS mapping.

In this study, we present a disposable and inexpensive paper-like gold nanoparticle-embedded cellulose nanofibril substrate for the rapid enumeration of Escherichia coli (E. coli) using surface-enhanced Raman scattering (SERS) mapping. A disposable SERS substrate was simply constructed by mixing CNF and gold chloride solution at 120 °C in a water bath. The application of the resulting substrate was carried out by enrichment and SERS detection of E. coli. To this end, the spherical gold nanoparticle-embedded cellulose nanofibril substrate was used as a scavenger for E. coli. After the target bacteria E. coli were separated from the matrix via oriented antibodies, the sandwich assay procedure was carried out using 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)-coated Au nanorod particles that acted as SERS mapping probes. The distribution density of DTNB was demonstrated visually using SERS mapping, and the assay was completed in one hour. The correlation between the E. coli and SERS mapping signals was found to be linear within the range of 15 cfu mL-1 to 1.5 × 105 cfu mL-1. The limit of detection for the SERS mapping assay was determined to be 2 cfu mL-1. The selectivity of the developed method was examined with Micrococcus luteus (M. luteus), Bacillus subtilis (B. subtilis), and Enterobacter aerogenes (E. aerogenes), which did not produce any significant response. Furthermore, the developed method was evaluated for detecting E. coli in artificially contaminated samples, and the results were compared with those of the plate-counting method.

Small numerosity advantage for sequential enumeration on RSVP stimuli: an object individuation-based account.

Although there is a large literature demonstrating rapid and accurate enumeration of small sets of simultaneously presented items (i.e., subitizing), it is unclear whether this small numerosity advantage (SNA) can also manifest in sequential enumeration. The present study thus has two aims: to establish a robust processing advantage for small numerosities during sequential enumeration using a rapid serial visual presentation (RSVP) paradigm, and to examine the underlying mechanism for a SNA in sequential enumeration. The results indicate that a small set of items presented in fast sequences can be enumerated accurately with a high precision and a SOA (stimulus onset asynchrony)-sensitive capacity limit, essentially generalizing the large literature on small numerosity advantage from spatial domain to temporal domain. A resource competition hypothesis was proposed and confirmed in further experiments. Specifically, sequential enumeration and other cognitive process, such as visual working memory (VWM), compete for a shared resource of object individuation by which items are segregated as individual entities. These results implied that the limited resource of object individuation can be allocated within time windows of flexible temporal scales during simultaneous and sequential enumerations. Taken together, the present study calls for attention to the dynamic aspect of the enumeration process and highlights the pivotal role of object individuation in underlying a wide range of mental operations, such as enumeration and VWM.