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Verma, M. S.

Publications and source records attributed to Verma, M. S..

18 recordsLinked to original sources

A rapid, field-deployable paper-based biosensor for the detection of African swine fever virus in whole blood

African swine fever virus (ASFV) poses a major transboundary threat to global swine production, underscoring the need for rapid and field-deployable diagnostic tools. Although quantitative polymerase chain reaction (qPCR)-based assays are the standard molecular assay for ASFV detection, their reliance on centralized laboratory infrastructure, multi-step sample preparation, and trained personnel limit their utility for timely decision-making at the point of need (PON). Here, we report a portable molecular diagnostic platform that enables colorimetric quantitative loop-mediated isothermal amplification (qLAMP) directly from diluted whole blood on microfluidic paper-based analytical devices ({micro}PADs). The assay targets the conserved ASFV viral protein 72 (VP72) and topoisomerase II (TOPII) genes and incorporates objective image-based colorimetric signal analysis to reduce user-dependent interpretation. Using plasmid DNA spiked into whole blood diluted to 5% (v/v) in 5% D-mannitol, the {micro}PAD-LAMP assay achieved a limit of detection (LOD) of 25 copies per reaction (67 copies/{micro}L of whole blood sample) for VP72 targets with no observed cross-reactivity against nine common swine pathogens, demonstrating 100% analytical sensitivity and specificity during in-house testing and 90% and 92% analytical sensitivity and specificity respectively in an external laboratory evaluation. The complete assay was performed within 60 minutes using a portable heating and imaging platform. Together, these results demonstrate a simple, DNA extraction-free molecular diagnostic approach that enables rapid and reliable ASFV detection from whole blood applicable to field-relevant conditions.

bioengineering↗

Bacteroidales on Harvesters: Baseline Prevalence and Abundance

Fresh produce encounters pathogens at various stages of production and supply, with the harvesting process serving as one of these stages. To evaluate contamination associated with harvesting, we systematically swabbed zone 1 harvester surfaces and quantified Bacteroidales as a fecal biomarker using quantitative polymerase chain reaction (qPCR). Baseline contamination was dominated by non-detects, with occasional low-level detections (<25 copies/cm2) near the assay limit of detection (LoD). Detection occurred more frequently post-harvest (overall [~]4% pre-harvest and 10% post-harvest), while microbial loads remained low, indicating that harvesting primarily affected the likelihood of low-level contamination rather than increasing contamination abundance. Additionally, we developed and field-deployed a portable loop- mediated isothermal amplification (LAMP) assay for rapid harvester hygiene assessment and benchmarked its field performance against qPCR. Together, these results support a practical molecular tool for monitoring fecal contamination and informing cleaning and sanitization decisions.

bioengineering↗

A solid-state heater-imager for quantitative evaluation of colorimetric isothermal nucleic acid amplification on paper

Maintaining precise isothermal conditions in portable nucleic acid amplification tests (NAATs) is critical for reproducible results but remains challenging with conventional single-sided thin-film heaters, which exhibit temperature gradients and strong dependence on ambient conditions. To close this gap, we engineered ThermiQuant VitroMini, a dual-sided heater design that achieves volumetric-level temperature uniformity using thin-film heaters while preserving optical transparency for real-time colorimetric loop-mediated isothermal amplification (LAMP) analysis on microfluidic paper-based analytical devices ({micro}PADs). The device integrates two independently regulated indium tin oxide (ITO) heaters (8 {Omega} each) controlled by independent proportional-integral-derivative (PID) algorithms. Heaters were evaluated under controlled ambient environments of 4 {degrees}C (refrigerated), 23 {degrees}C (room temperature), and 50 {degrees}C (oven). Analytical tests were performed using a colorimetric LAMP assay targeting the SARS-CoV-2 orf7ab gene on {micro}PADs preloaded with dried LAMP reagents, with time-lapse images (30 seconds interval) analyzed via Amplimetrics software. VitroMini maintained 65 {+/-} 0.5 {degrees}C across 4 to 50 {degrees}C ambient conditions and achieved a limit of detection of 34 copies/reaction (4.5 copies/{micro}L) and limit of quantification of 1000 copies/reaction (133 copies/{micro}L), with quantification time (Tq) linearly correlated with log10 DNA concentration. Dual-sided heating eliminated temperature bias, condensation artifacts, and ambient-dependent variability while preserving optical transparency for real-time quantitative LAMP reaction. ThermiQuant VitroMini bridges the gap between benchtop volumetric heaters and portable diagnostic devices, offering a compact, low-power platform for quantitative colorimetric molecular analysis on paper with potential for decentralized and field-deployable applications.

bioengineering↗

ThermiQuantTM AquaStream: A Portable Instrument for Quantitative Colorimetric Isothermal Nucleic Acid Amplification Reactions in Paper and Tube Formats

Microfluidic paper-based analytical devices ({micro}PADs) are an attractive format for colorimetric nucleic acid amplification tests (NAATs) because they enable low-cost, portable diagnostics in resource-limited settings. However, researchers often optimize colorimetric assays in liquid reactions in tubes before translating them to {micro}PADs. Since both formats require separate instruments for incubation and real-time sensing, direct comparison of reactions between the two formats is difficult. To address these cross-platform limitations, we developed ThermiQuant AquaStream, a portable benchtop device (15 x 20 x 16 cm, [~]5 kg; cost: USD 327) that supports seamless colorimetric loop-mediated isothermal amplification (LAMP) reactions in both {micro}PADs and tubes under a common workflow. The system enables real-time reaction tracking (every 30 seconds) through onboard image processing, precise isothermal control ({+/-}0.5 {degrees}C) using a repurposed consumer-grade sous-vide heater, and medium-throughput (24 tubes or 42 {micro}PADs). Testing with synthetic SARS-CoV-2 orf7ab DNA fragments demonstrated a limit of detection corresponding to a 95% probability of detection (LOD95) of 110 copies per reaction in tube (22 copies/{micro}L) and 39 copies per reaction in {micro}PADs (5 copies/{micro}L), estimated using probit regression. In both formats the limit of quantification (LOQ), defined as the lowest concentration yielding a coefficient of variation (CV) of Tq [&le;] 10%, was 250 copies/reaction resulting in a strong linear (R2 = 0.98 & R2 = 0.96 respectively for tube and {micro}PADs) standard calibration curves. ThermiQuant AquaStream provides an affordable and versatile benchtop platform capable of supporting both tube- and {micro}PAD-based colorimetric LAMP assays, serving as a proof-of-concept research tool for assay development and molecular diagnostics in One Health settings such as clinics, farms, and field environments.

bioengineering↗

ThermiQuant(TM) MegaScan: High-throughput isothermal reactor with quantitative colorimetric readout for paper-based nucleic acid amplification tests

Isothermal nucleic acid amplification tests (NAATs), such as loop-mediated isothermal amplification (LAMP) implemented on microfluidic paper-based analytical devices ({micro}PADs), enable inexpensive and rapid ([&le;]60 min) colorimetric molecular diagnostics; however, no existing instrument supports high-throughput (>100 reactions) quantitative analysis of colorimetric isothermal assays on paper substrates under controlled laboratory conditions. To address this gap, we developed ThermiQuant MegaScan, a scanner- and water-bath-based platform that accommodates a 160-reaction {micro}PAD cartridge, maintains uniform incubation at 65 {+/-} 0.5 {degrees}C, and enables real-time imaging every 30 s. We also developed accompanying software, Amplimetrics, for automated {micro}PAD detection and kinetic colorimetric analysis. Using paper-based colorimetric LAMP targeting the SARS-CoV-2 orf7ab region, the assay achieved a limit of detection at 95% probability (LOD95) of 34 copies per reaction (5 copies/{micro}L) and a limit of quantification (LOQ) of 250 copies per reaction (33 copies/{micro}L) using purified synthetic DNA targets, and achieved 72% sensitivity and 100% specificity relative to digital PCR (dPCR) for diluted human nasopharyngeal (NP) swab virus samples. We further evaluated the effects of viral and universal transport media (VTM/UTM) on assay performance and found that linear calibration derived from synthetic targets do not reliably translate to clinical samples in these media. Together, these results establish ThermiQuant MegaScan as a high-throughput laboratory research platform for standardized evaluation, optimization, and benchmarking of paper-based colorimetric nucleic acid amplification assays.

bioengineering↗

Detection of five viruses commonly implicated with Bovine Respiratory Disease using loop-mediated isothermal amplification

Herein, we present novel quantitative loop-mediated isothermal amplification (qLAMP) and reverse-transcription qLAMP (RT-qLAMP) assays for the detection of five viruses commonly implicated with the onset and progression of bovine respiratory disease (BRD): Bovine Alphaherpesvirus Type 1 (BHV-1), Bovine Adenovirus Type 3 (BAV-3), Bovine Respiratory Syncytial Virus (BRSV), Bovine Viral Diarrhea Virus Type 1 (BVDV-1), and Bovine Parainfluenza Virus Type 3 (BPIV-3). Using contrived samples spiked with whole viruses, we found that our extraction-free assays have limits of detection between 30 and 1,057 copies per reaction (1.8% final sample concentration) with minimal sample processing. Using dual-tipped swabs and 1.4 mL resuspension volumes, these limits of detection are on the order of 2 x 105 copies per swab for BAV-3 and BHV-1 and between 6.31 x 106 to 8.22 x 106 copies per swab in the case of BPIV-3, BRSV, and BVDV-1. Analytical sensitivities ranged from 73 - 100% and analytical specificities ranged from 90 - 100%. Additionally, we introduced a streamlined pipeline to minimize the experimental workload to design, screen, select, and characterize LAMP performance for developing assays. The assays targeting these BRD viruses can be utilized to develop colorimetric LAMP assays that enable the sensitive and specific detection of these viruses chute side to aid in diagnosing and treating BRD. The associated development pipeline enables more rapid development of LAMP-based diagnostic tools targeting emerging pathogens.

bioengineering↗

Development and Evaluation of Novel Quantitative PCR (qPCR) and Loop-Mediated Isothermal Amplification (LAMP) Assays for Bovine Adenovirus Type 7

Herein, we report the development of molecular assays for the specific detection of bovine adenovirus type 7 (BAV-7), a prevalent pathogen associated with bovine respiratory disease (BRD). To overcome the limitations of the current diagnostic methods, we developed and optimized a TaqMan quantitative polymerase chain reaction (qPCR) assay with a limit of detection (LOD) of 50 copies per reaction (10 copies/{micro}L) and 100% analytical specificity, showing no cross-reactivity with nine other viral pathogens commonly linked to BRD. Additionally, we designed a fluorescent quantitative loop-mediated isothermal amplification (qLAMP) assay demonstrating equivalent LOD and specificity. Both assays were evaluated using 24 bovine nasal swabs from BRD animals, revealing a 92% agreement between qPCR and qLAMP results. These assays provide powerful tools for advancing our understanding of BAV-7 epidemiology and improving disease management strategies. The qLAMP assay, with its rapid and isothermal amplification, presents potential for future development as a field-deployable diagnostic tool to enable timely detection of BAV-7 in cattle.

bioengineering↗

Evaluation of Paper-based Loop-Mediated Isothermal Amplification

Colorimetric loop-mediated isothermal amplification (LAMP) on microfluidic paper-based analytical devices (PADs) offers a low-cost, disposable, and equipment-free alternative to liquid LAMP assays. However, amplification on PADs is consistently slower, by 5-46%, than reactions in tubes. To identify the origin of this delay, we evaluated heat transfer, diffusion in porous cellulose, and nonspecific adsorption of LAMP components across both high- and low-copy input regimes. Our results show that once thermal equilibrium is reached, reduced effective diffusion is the dominant contributor to the kinetic lag at low copy numbers, whereas nonspecific adsorption becomes the primary barrier at higher template concentrations. Pre-coating the paper with bovine serum albumin (BSA) mitigates adsorption. It narrows the tube-to-paper gap, thereby accelerating amplification of the SARS-CoV-2 ORF7ab synthetic gene by an average of 6 minutes, from 1E3 to 1E5 copies per reaction. These findings provide a mechanistic basis for the copy-number-dependent behavior of PAD LAMP and offer simple, low-cost strategies to improve the speed and reliability of PAD nucleic acid assays.

bioengineering↗

Limit of detection of Raman spectroscopy using polystyrene particles from 25 to 1000 nm in aqueous suspensions

Raman spectroscopy is an analytical method capable of detecting various microorganisms and small particles. Here, we used 25-1000 nm polystyrene particles in aqueous suspensions, which are comparable in size to viral particles and viral aggregates, to determine the limit of detection of a confocal Raman microscope. We collected Raman spectra using a 785 nm wavelength laser with a power of 300 mW and a 10 s exposure time, with a 5X objective lens. We detected the most prominent peak of the polystyrene particles at 1001 cm-1, corresponding to the ring breathing mode. We established the minimum and maximum limit of detection (LODmin and LODmax) using a partial least squares (PLS) model. The LOD of the smallest size of 50 nm was identified as 7.47 x 1012 -7.64 x 1012 particle/mL, and for the largest size of 1000 nm, 6.09 x 108 -6.24 x 108 particle/mL. We demonstrated that Raman spectroscopy was non-destructive under these conditions by comparing the particle size before and after collecting Raman spectra using dynamic light scattering. Due to their size similarity to viral particles and viral aggregates, this systematic characterization of polystyrene particles provides detailed information on their Raman spectral signatures in aqueous suspensions. These findings establish a foundation for using Raman spectroscopy for the detection of small particles in aqueous suspensions and highlight its potential as a tool for real-time monitoring in vaccine manufacturing.

bioengineering↗

A Paper-based Loop-Mediated Isothermal Amplification (LAMP) Assay for Highly Pathogenic Avian Influenza

Avian influenza outbreaks have had significant economic and public health consequences worldwide. Therefore, prompt, reliable, and cost-effective diagnostic devices are crucial for scrutinizing and confining highly pathogenic avian influenza viruses (HPAIVs). Our study introduced and evaluated a novel paper-based loop-mediated isothermal amplification (LAMP) test for diagnosing the H5 subtype of the avian influenza virus (AIV). We meticulously designed and screened LAMP primers targeting the H5-haemagglutinin (H5-HA) gene of AIV and fine-tuned the paper-based detection assay for best performance. The paper-based LAMP assay demonstrated a detection limit of 500 copies per reaction (25 copies/{micro}L). This inexpensive, user-friendly point-of-need diagnostic tool holds great promise, especially in resource-limited settings. It only requires a water bath for incubation and enables visual detection of results without special equipment. Overall, the paper-based LAMP assay provides a promising method for rapidly and reliably detecting the H5 subtype of AIV, contributing to improved surveillance and early intervention strategies.

bioengineering↗

Propidium monoazide is unreliable for quantitative live-dead molecular assays

Propidium monoazide (PMA) is a dye that distinguishes between live and dead cells in molecular assays like Polymerase Chain Reaction (PCR). It works by cross-linking to the DNA of cells that have compromised membranes or extracellular DNA upon photoactivation, making the DNA inaccessible for amplification. Currently, PMA is used to detect viable pathogens and alleviate systemic bias in the microbiome analysis of samples using 16S rRNA gene sequencing. In these applications, treated samples consist of different amounts of dead bacteria and a range of bacterial strains, variables that can affect the performance of PMA and lead to inconsistent findings across various research studies. To evaluate the effectiveness of PMA, we used a sensitive qPCR assay and post-treatment sample concentration to determine PMA activity accurately under varying sample conditions. We report that PMA is unreliable for viability assays when the concentration and composition of the bacterial mixture are unknown. PMA is only suitable for qualitatively assessing viability in samples containing a known number of dead microbes or extracellular DNA.

bioengineering↗

Design and development of a field-deployable water bath for loop-mediated isothermal amplification assay

Nucleic acid testing has become a prominent method for rapid microbial detection. Unlike polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) is a simple method of nucleic acid amplification where the reaction can be performed at a constant temperature and the output provided in a colorimetric format. A transparent water bath is a desirable instrument to perform the heating and observe the visual results. However, existing methods of heating water are not convenient for loading and unloading the test samples. Here, we developed a field-deployable water bath--an isothermal heater called IsoHeat for short-which is solely dedicated to performing LAMP reactions and can heat the water up to 85{degrees}C. Using 3D-printing and laser-cutting technology, we fabricated different parts and mechanically assembled the parts to develop the entire device. Users can commence the heating by pressing the start button on the screen after entering the target temperature. Subsequently, the device heats up the water bath and maintains the target temperature through a PID algorithm-based control system. We demonstrate that IsoHeat can operate in environmental temperatures ranging from 5-33 {degrees}C and it can conduct LAMP reactions in liquid format as well as in paper-based devices. IsoHeat is more efficient and user-friendly compared to a commercially available immersion-heating device, which is often used to perform LAMP reactions. This newly developed device would be helpful to detect pathogens conveniently in the field (e.g., at point-of-care for human applications, on farms for plant and animal applications, and in production facilities for food safety applications).

bioengineering↗

Real-time monitoring of attenuated cytomegalovirus using Raman spectroscopy allows non-destructive characterization during flow

Real-time monitoring of viral particles can have a crucial impact on vaccine manufacturing and can alleviate public health by supporting continuous supply. Spectroscopic methods such as Raman spectroscopy can provide rapid and non-invasive measurements. Here, we have developed a Raman spectroscopy-based tool to monitor the quality and quantity of viral particles in a continuous flow set-up. The attenuated human cytomegalovirus (CMV) is characterized across a wide range of concentrations (4.50 x 109 to 2.90 x 1011 particles/mL) and flow rates (100 {micro}m/s to 1000 {micro}m/s) within a square quartz capillary. This process analytical technology (PAT) tool enables the detection of viral particles even at high flow rates such as 1000 {micro}m/s. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) demonstrated that the samples maintain their integrity even after laser exposure, reiterating the non-invasive nature of Raman spectroscopy. To the best of our knowledge, this is the first report on characterizing CMV particles using Raman spectroscopy. We have also demonstrated the limit of detection (LoD) (2.36 x 1010 particles/mL) for CMV particles in continuous flow (via the Raman spectroscopy method) by addressing the effect of flow rate, concentration, and integrity of samples. This technology could enhance our understanding of the quality control in bio-manufacturing processes required in vaccine production.

bioengineering↗

Super-resolved protein imaging using bifunctional light-up aptamers

Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single-molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light-up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super-resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein-binding aptamers. The versatility of such bifunctional aptamers is demonstrated by employing a variety of protein-binding DNA or RNA aptamers and different FLAPs. Moreover, fusing RhoBAST with the GFP-binding aptamer AP3 facilitates high- and super-resolution imaging of GFP-tagged proteins, which is particularly valuable in view of the widespread availability of plasmids and stable cell lines expressing proteins fused to GFP. The bifunctional aptamers compare favorably with standard antibody-based immunofluorescence protocols, as they are 7-fold smaller than antibody conjugates and exhibit higher bleaching-resistance. We demonstrate the effectiveness of our approach in super-resolution microscopy in secondary mammalian cell lines and primary neurons by RhoBAST-PAINT, an SMLM protein imaging technique that leverages the transient binding of the fluorogenic rhodamine dye SpyRho to RhoBAST.

biophysics↗

A portable, easy-to-use paper-based biosensor for rapid in-field detection of fecal contamination on fresh produce farms

Laboratory-based nucleic acid amplification tests (NAATs) are highly sensitive and specific, but they require the transportation of samples to centralized testing facilities and have long turnaround times. During the Coronavirus Disease 2019 (COVID-19) pandemic, substantial advancement has been achieved with the development of paper-based point-of-care (POC) NAATs, offering features such as low cost, being easy to use, and providing rapid sample-to-answer times. Although most of the POC NAATs innovations target clinical settings, we have developed a portable, paper-based loop-mediated isothermal amplification (LAMP) testing platform for on-farm applications, capable of detecting Bacteroidales as a fecal contamination biomarker. Our integrated platform includes a drop generator, a heating and imaging unit, and paper-based biosensors, providing sensitive results (limit of detection 3 copies of Bacteroidales per cm2) within an hour of sample collection. We evaluated this integrated platform on a commercial lettuce farm with a concordance of 100% when compared to lab-based tests. Our integrated paper-based LAMP testing platform holds great promise as a reliable and convenient tool for on-site NAATs. We expect that this innovation will encourage the fresh produce industry to adopt NAATs as a complementary tool for decision-making in growing and harvesting. We also hope that our work can stimulate further research in the development of on-farm diagnostic tools for other agricultural applications, leading to improved food safety and technology innovation.

bioengineering↗

Bacteroidales as a Fecal Contamination Indicator in Fresh Produce Industry: A Baseline Measurement

Foodborne outbreaks caused by fecal contamination of fresh produce represent a serious concern to public health and the economy. As the consumption of fresh produce increases, public health officials and organizations have pushed for improvements in food safety procedures and environmental assessments to reduce the risk of contamination. Visual inspections and the establishment of "buffer zones" between animal feeding operations and producing fields are the current best practices for environmental assessments. However, a generalized distance guideline and visual inspections may not be enough to account for all environmental risk variables. Here, we report a baseline measurement surveying the background Bacteroidales concentration, as a quantitative fecal contamination indicator, in Californias Salinas Valley. We collected a total of 1632 samples from two romaine lettuce commercial fields at the time of harvesting through two seasons in a year. The Bacteroidales concentration was very low (0 - 2.00 copies/cm2). Furthermore, we established a practical methodology for evaluating the risk of fecal contamination in a real-world setting, complementing the current environmental assessment practices. This method can identify site-specific risks and offer fresh produce stakeholders a more comprehensive understanding of their fields. We anticipate this work can encourage the use of Bacteroidales in the fresh produce industry to monitor fecal contamination and prevent future foodborne outbreaks.

bioengineering↗

A drop dispenser for simplifying on-farm detection of foodborne pathogens

Rapid nucleic-acid biosensors are useful for on-farm detection of foodborne pathogens on fresh produce during pre-season and pre-harvest stages. Such tools aim to be user-friendly so that a producer could operate them in a few simple steps and detect multiple targets. Currently, an easy-to-use device for on-farm applications does not exist commercially. One of the bottlenecks is the delivery of a prescribed amount of sample to the reaction sites of the biosensor using a simple and precise approach. Here, we developed drop dispensers using 3D printing and a hydrophilic surface chemistry treatment to generate precise drops (DNA/bacterial samples) of a few micro-liters ([~]20 to [~]33 {micro}L). We tested multiple copies of these devices over time of repeated application to estimate their shelf-lives which is about one month. In addition to drop generation tests, we employed these devices in nucleic-acid testing. The tests used loop-mediated isothermal amplification (LAMP) to detect DNA or whole cells of Shiga-toxin-producing Escherichia coli O157:H7. These tests were performed to simulate the on-farm sample collection (using collection flags that we previously designed) and delivery using the drop dispensers. Our results showed that these devices performed similarly to standard commercial pipettors in LAMP assays, providing a limit of detection of 7.8x106 cell/mL for whole-cell detection. This drop dispenser will eventually be part of a user-friendly consumable kit that will enable performing LAMP assays by non-specialist users for a cost of USD 4 per test.

bioengineering↗

Modelling complex growth profiles of Bacteroides fragilis and Escherichia coli on various carbohydrates in an anaerobic environment

Previously published models for microbial growth focus only on either death or growth and are unable to account for differently shaped growth curves. Currently, there is no model capable of incorporating combinations of microbial growth trends. This study creates a bacterial growth model that incorporates growth, death, lag, and tail phases as well as applies this model to the growth trends of Bacteroides fragilis and Escherichia coli on 13 different carbohydrate substances. Growth trends were collected by measuring the optical densities over 72 hours for either B. fragilis or E. coli in a chemically defined media supplemented by a mono- or disaccharide. The Digital Environment to Enable Data-driven Science (DEEDS) platform was utilized to parse data and apply the developed model to obtain parameter values. E. coli was found to grow on the chemically defined media alone while B. fragilis was unable to grow on it alone. E. coli growth was led by 10 mM concentration of substrates while B. fragilis growth was substrate dependent. Bacterial death only occurred for B. fragilis but was found to be dependent on concentration for the two most significant substrates. A singular model was developed that does not require prior knowledge of metabolomics and is capable of incorporating a combination of growth and death trends.

microbiology↗