OnePetri: accelerating common bacteriophage Petri dish assays with computer vision

IntroductionBacteriophage 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. This approach is time-intensive, has low-throughput, is limited to Petri dishes which have a countable number of plaques, and can have variable results upon recount due to human error.MethodsWe 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.ResultsWhen compared against the current gold standard of manual counting, OnePetri was significantly faster, with minimal error. Compared against two other similar tools, Plaque Size Tool and CFU.AI, OnePetri had higher plaque recall and reduced detection times on most test images.ConclusionsThe OnePetri application can rapidly enumerate phage plaques on circular Petri dishes with high precision and recall.

Reverse Transcriptase Real-Time PCR Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcriptase (RT) real-time PCR assay for rapid detection of live C. auris in healthcare environments. The assay targets the internal transcribed spacer 2 (ITS2) cDNA of C. auris, amplified by obtaining pure RNA followed by reverse transcription and real-time PCR assay. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT real-time PCR reaction. In C. auris viability studies, ITS2 cDNA was detectable in heat- and ethanol-killed C. auris cells, but not bleach-killed cells. Validation studies showed no cycle threshold (Ct) values were obtained from samples on a sponge matrix spiked with either dead C. auris (105/ml) or other Candida species (105/ml), while most environmental samples spiked with 102 to 105 CFU of live C. auris yielded positive Ct values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT real-time PCR assay, confirming concordance between culture and the assay. The RT real-time PCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples, and could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

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

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.

Effectiveness of Nitrogen Dioxide Fumigation for Microbial Control on Stored Almonds

ABSTRACT 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. HIGHLIGHTS

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 highly efficient, low-cost, novel multicomponent nanosystem for rapid enumeration of circulating tumor cells.

e14516 Background: ‘Liquid biopsy’ technologies are unaffordable and unavailable in developing countries despite having highest cancer burden and mortality rates. Current Circulating Tumor Cell (CTCs) technologies sustain clinical concerns of a) non-specificity b) low efficiency c) high blood volume requirement d) long turn-around time, and d) exorbitant cost (~$900-1400). We report, an extremely low cost, innovative nanosystem for rapid enumeration of CTS with higher specificity and efficiency. Methods: We designed a nanosystem mediated by conjugation of anti-EpCAM through multi-reactive glutathione spacer, carbon allotrope and amine terminated dendrimer. The platform was evaluated for enhanced aqueous dispersibility and increased interaction with CTCs for rapid isolation and enumeration in 100 Head and Neck Cancer (HNC) patients having primary tumor sub-sites-oral cavity, larynx, hypopharynx, oropharynx, nasopharynx, salivary gland, and thyroid. The captured cells were immuno-stained and the optimal fluorescence acquisition intensity was validated in accounting CTCs with CK18 protein expression. There was complete elimination of the false positive normal cells (NC) count to CTCs by our method. The analysis was performed with only 1.5 ml of collected blood samples. Results: The CTC distribution in cohort study ranged from 1 - 85 cells per 1.5 mL of blood. In more than 80% of patient’s CTCs, the quantitative estimation of anti-CK18 protein over-expression indicated ~10-fold higher intensity over to NCs. As compared to treatment naive, recurrent, and disease-free patients, the spread of CTC number across the clinical range appeared to be tight (close to mean value). The CTC enumeration sensitivity linearity was ~99.2%, and the complete enumeration process time was under 03 hours/1.5 ml of blood. Consequently, efficient, rapid and yet affordable CTC platform was designed and clinically validated. Conclusions: ‘OncoDiscover’ liquid biopsy technology for CTC enumeration is poised to revolutionize the liquid biopsy due to its high sensitivity and affordability (~ $120) and shall resolve a major unmet medical need in impoverished world.

Whole slide imaging of circulating tumor cells captured on a capillary microchannel device

The integrated capillary microchannel modified with polymer brushes allows direct blood sampling, efficient CTC capturing, and rapid enumeration with whole slide imaging.

Ultra-High-Throughput Multi-Parametric Imaging Flow Cytometry

I will present a microfluidic imaging flow cytometer incorporating stroboscopic illumination, for blur-free cellular analysis at throughputs exceeding 100,000 cells per second. By combining passive (inertial or viscoelastic) focusing of cells in parallel microchannels with stroboscopic illumination, such chip-based cytometers are able to extract multi-colour fluorescence and bright-field images of single cells moving at high linear velocities. This in turn allows accurate sizing of individual cells, intracellular localization and analysis of heterogeneous cell suspensions. The method is showcased through the rapid enumeration of apoptotic cells, high-throughput discrimination cell cycle phases and localization of p-bodies.