Life Science Laboratories Research Articles

Bio-based polylactic acid labware as a sustainable alternative for microbial cultivation in life science laboratories

Single-use plastics (SUPs) in life science laboratories account for approximately 5.5 million tonnes of waste per year globally. Of SUPs used in life science laboratories, Petri dishes, centrifuge tubes, and inoculation loops are some of the most common. In order to reduce the reliance on petrochemical-based SUPs in the life science research laboratory and minimize the negative environmental impacts associated with SUPs, this research investigates the applicability of polylactic acid (PLA) in single-use labware as a replacement for petrochemical-based plastics. PLA is one of the most well-studied biodegradable plastics that can be produced from sustainable resources. Commercially available PLA was used to 3D print a select range of labware to test the suitability of PLA-based material for routine microbiology work. An injection moulded PLA-based Petri dish was also designed and produced, for increased optical clarity. The biocompatibility was tested against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus epidermidis) strains of bacteria. The PLA-based labware did not negatively impact the cell growth, viability, and metabolic activity of the bacterial cultures. The injection moulded PLA Petri dish showed a reduced colony forming unit count for the Gram-negative E. coli strain compared to the polystyrene Petri dish, ∼1.5 × 109 CFU/mL and ∼3.0 × 109 CFU/mL respectively, during late-exponential growth. The colony counts were, however, in the same order of magnitude. This observed difference may be due to the internal environment inside the Petri dish, hence the internal O2 concentration, humidity, and temperature during bacterial growth were investigated. This work demonstrates, for the first time, a full successful workflow of bacterial growth using a sustainable bioplastic, providing a pathway to reducing the environmental impacts of SUPs in life science laboratories.

Enhancing quantitative imaging to study DNA damage response: A guide to automated liquid handling and imaging

Laboratory automation and quantitative high-content imaging are pivotal in advancing diverse scientific fields. These innovative techniques alleviate the burden of manual labour, facilitating large-scale experiments characterized by exceptional reproducibility. Nonetheless, the seamless integration of such systems continues to pose a constant challenge in many laboratories. Here, we present a meticulously designed workflow that automates the immunofluorescence staining process, coupled with quantitative high-content imaging to study DNA damage signalling as an example. This is achieved by using an automatic liquid handling system for sample preparation. Additionally, we also offer practical recommendations aimed at ensuring the reproducibility and scalability of experimental outcomes. We illustrate the high level of efficiency and reproducibility achieved through the implementation of the liquid handling system but also addresses the associated challenges. Furthermore, we extend the discussion into critical aspects such as microscope selection, optimal objective choices, and considerations for high-content image acquisition. Our study streamlines the image analysis process, offering valuable recommendations for efficient computing resources and the integration of cutting-edge deep learning techniques. Emphasizing the paramount importance of robust data management systems aligned with the FAIR data principles, we provide practical insights into suitable storage options and effective data visualization techniques. Together, our work serves as a comprehensive guide for life science laboratories seeking to elevate their high-content quantitative imaging capabilities through the seamless integration of advanced laboratory automation.

Strategic naturalizing in the Anthropocene: Managing cells, bodies and ecosystems

Discussions on the Anthropocene have called for increased attention to how the effects of human actions on the planet are accounted for. While much of this debate remains at a theoretical level, more studies on situated Anthropocene realities have been called for. Contributing to the latter, this article explores how experimental and clinical interventions are being accounted for in life science laboratories. Drawing on three research cases, genetically modified mosquitoes, laboratory-grown stem cell lines and assisted reproduction in the IVF clinic, the article traces how ‘strategic naturalizing’ is conducted to make sense of and justify human interventions on cells, bodies and ecosystems. Strategic naturalizing in laboratory work is situated, fluid, and also implicates the presence of the social scientist. Although naturalizing biotechnologies is not a new phenomenon, according to our observations scientists increasingly draw on notions of a planet profoundly transformed by human intervention as they conceptualize their own accountability. Consequently, we propose that strategic naturalizing is emerging as an elemental logic in the patchy local enactments of the Anthropocene and its concerns. In the context of experimental and clinical laboratory work, it is a key element in enacting good science and orienting the work towards making biotechnology acceptable in wider society. In studying this, the social scientist also is implicated in situated local enactments of the Anthropocene.

Multimodal analysis of traction forces and the temperature dynamics of living cells with a diamond-embedded substrate.

Cells and tissues are constantly exposed to chemical and physical signals that regulate physiological and pathological processes. This study explores the integration of two biophysical methods: traction force microscopy (TFM) and optically detected magnetic resonance (ODMR) to concurrently assess cellular traction forces and the local relative temperature. We present a novel elastic substrate with embedded nitrogen-vacancy microdiamonds that facilitate ODMR-TFM measurements. Optimization efforts focused on minimizing sample illumination and experiment duration to mitigate biological perturbations. Our hybrid ODMR-TFM technique yields TFM maps and achieves approximately 1 K precision in relative temperature measurements. Our setup employs a simple wide-field fluorescence microscope with standard components, demonstrating the feasibility of the proposed technique in life science laboratories. By elucidating the physical aspects of cellular behavior beyond the existing methods, this approach opens avenues for a deeper understanding of cellular processes and may inspire the development of diverse biomedical applications.

SARS-CoV-2 Catastrophe and aftermath in 3800 Indian patients with comorbidities: A retrospective study

Introduction: The severity of SARS-CoV-2 infections on the basis of hospitalization, ventilation support, oxygen therapy, and morbidity has been a major challenge witnessed by the medical fraternity during 2020 due to the global pandemic. The correlation and effects of the severity caused by the disease outcome due to SARS-CoV-2 infections along with co-morbid factors and lifestyle habits in the diverse Indian population are of greater interest to evaluate. Objective: In this study, we aimed to investigate the association between various co-morbid factors, addiction habits, and their contribution towards disease severity, and the need for hospitalisation in a retrospective manner in a cohort of SARS-CoV-2 positive patients from the eastern part of India. Methodology: Swab samples from a total of 75000 individuals were screened by RT-PCR technology between the months of July-November, 2020 at the inDNA Life Sciences laboratory as per ICMR guidelines. Out of 75000 individuals, 3800 SARS-CoV-2 positive individuals were included in this study. Retrospectively, the positive cases were considered for follow-up investigations. Clinical correlation between the severity of the disease and co-morbid factors along with addiction habits were performed. Results: We observed that the young age group of 20-30 had the highest incidence of SARS-CoV-2 and the majority of infected individuals were males (66%). Disease transmission was high with the majority of asymptomatic patients presenting with low-grade fever. It was also observed that disease severity, mortality, and average recovery time (>14 days) were higher in patients with co-morbid conditions as compared to patients without co-morbidity. In addition, patients with a history of addiction exhibited higher severity of the disease and longer hospitalization with comparatively high mortality. Conclusion: In this retrospective study we found, that co-morbid conditions such as diabetes, hypertension, COPD, asthma, liver disorder, and kidney disorder contributed to the severity or had detrimental outcomes of SARS-CoV-2 infections. Addiction to alcohol and smoking/chewing tobacco also results in poor clinical outcomes of the disease. Therefore, understanding the relationship between these risk factors and their association with disease outcomes can further assist the medical fraternity in managing the current patients in the clinic/ hospitals with a prior history of SARS-CoV-2 infection. Years after the outbreak, COVID-19 still remains a gray area with probably a lot of answers to be given to mankind.

Evaluation of Chemical Composition, Physicochemical and Anti-nutritional Properties of Giant Yellow Mulberry Fruit (Myrianthus arboreus)

The study aimed at evaluating the nutritional value of the pulp and seeds of the giant mulberry (Myrianthus arboreus) fruit, and determine the physicochemical properties of its seed oil. Using the complete randomized design, mature ripe fruits of the fruits were harvested from Fontem, South west region, Cameroon and analyzed at the life science laboratory of the University of Buea following standard procedures. Results revealed the proximate composition of the pulps and seeds were 5.03% and 28.06% for carbohydrates, 17.22% and 24.40% for proteins, 6.50% and 42.05% for lipids respectively. Antinutritional analyses revealed moderately low content of phytate, tannin and oxalate. The oil quality indices demonstrated acceptable values for acid, saponification value, iodine, peroxide and P-anisidine. Fatty acid profiling showed that the oil was 45.06% saturated and 54.94% unsaturated fatty acids accounts. These findings suggest that M. arboreus fruit as potentials as a nutritious food source with health benefits.

Silver Nanoparticles and Graphene Oxide Complex as an Anti-Inflammatory Biocompatible Liquid Nano-Dressing for Skin Infected with Staphylococcus aureus.

Bacterial skin infections, including Staphylococcus aureus, are a powerful and still not fully resolved problem. The aim of this research was to determine the possibility of using a complex of graphene oxide (GO) encrusted with silver nanoparticles as an effective antibacterial agent against S. aureus and to assess its pro-inflammatory properties. The tests were carried out in vitro on EpiDerm™ Skin, an artificial skin model (MatTek in vitro Life Science Laboratories, Slovak Republic), and the fibroblast cell line (HFF-2 from ATCC, USA). Both models were infected with S. aureus bacteria (ATCC 25923) and then treated with antibiotics or our experimental factors: silver nanoparticles (AgNPs, Nano-koloid, Poland), graphene oxide (GO, NanoPoz, Poland), and complex AgNP-GO (hydrocolloid created by self-assembly). The antibacterial effectiveness of the AgNP-GO complex was equivalent to that of the antibiotic. In addition, an increase in the level of pro-inflammatory cytokines was observed under the influence of antibiotic administration, in contrast to the effect of AgNP-GO, which showed very limited pro-inflammatory activity. Hydrocolloid of the AgNP-GO complex, administered in the form of a liquid dressing, may act as an antibacterial agent and also reduce inflammation induced by S. aureus infection.

REVIEW: AN APPROACH TO POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle, high resolution method for fractionation and physical-chemical characterization of molecules on the basis of size, conformation, and net charge. It is a technique used almost universally in life science laboratories. The goal of this technique is to separate a mixed sample of proteins, to identify and quantify single proteins from the mixture. It is also used to separate DNA and RNA, although proteins remain the most common sample type. It can also be used to resolve RNA protein complexes and to detect RNA complex formation by analyzing changes in the electrophoretic mobility of the RNA. Major advantage of PAGE is its high resolving power (sometimes 1 in 500 bp).

Open Hardware for Microfluidics: Exploiting Raspberry Pi Singleboard Computer and Camera Systems for Customisable Laboratory Instrumentation.

The integration of Raspberry Pi miniature computer systems with microfluidics has revolutionised the development of low-cost and customizable analytical systems in life science laboratories. This review explores the applications of Raspberry Pi in microfluidics, with a focus on imaging, including microscopy and automated image capture. By leveraging the low cost, flexibility and accessibility of Raspberry Pi components, high-resolution imaging and analysis have been achieved in direct mammalian and bacterial cellular imaging and a plethora of image-based biochemical and molecular assays, from immunoassays, through microbial growth, to nucleic acid methods such as real-time-qPCR. The control of image capture permitted by Raspberry Pi hardware can also be combined with onboard image analysis. Open-source hardware offers an opportunity to develop complex laboratory instrumentation systems at a fraction of the cost of commercial equipment and, importantly, offers an opportunity for complete customisation to meet the users' needs. However, these benefits come with a trade-off: challenges remain for those wishing to incorporate open-source hardware equipment in their own work, including requirements for construction and operator skill, the need for good documentation and the availability of rapid prototyping such as 3D printing plus other components. These advances in open-source hardware have the potential to improve the efficiency, accessibility, and cost-effectiveness of microfluidic-based experiments and applications.

Immunocytochemical Visualization of Proteins from Cyanobacterial Cells with High Autofluorescence of Phycoerythrin and Phycourobilin.

Presented is a protocol for visualizing and quantifying a specific protein in cells at the cellular level for the marine cyanobacterium Crocosphaera watsonii, a crucial primary producer and nitrogen fixer in oligotrophic oceans. One of the challenges for marine autotrophic N2 fixers (diazotrophs) is distinguishing probe-derived fluorescence signals from autofluorescence. C. watsonii was selected to represent chlorophyll-, phycoerythrin- and phycourobilin-containing cyanobacteria. The protocol allows for simple and semi-quantitative visualization of proteins in C. watsonii at a single-cell level, enabling investigation of protein production under different environmental conditions to evaluate the metabolic activities of the target cyanobacteria. Furthermore, the fixation and permeabilization methods are optimized to enhance the fluorescence signals from target proteins to distinguish them from autofluorescence, especially from phycoerythrin and phycourobilin. The enhanced signal can be visualized using confocal or widefield fluorescence microscopy. Additionally, fluorescence intensity was semi-quantified using Fiji software. This single-cell analysis workflow allows the evaluation of cell-to-cell variations of specific protein content. The protocol can be performed in any life science laboratory as it requires only standard equipment and can also be easily adapted to other phycoerythrin-containing cyanobacterial cells.

Towards robotic laboratory automation Plug & play: Survey and concept proposal on teaching-free robot integration with the lapp digital twin.

The Laboratory Automation Plug & Play (LAPP) framework is an over-arching reference architecture concept for the integration of robots in life science laboratories. The plug & play nature lies in the fact that manual configuration is not required, including the teaching of the robots. In this paper a digital twin (DT) based concept is proposed that outlines the types of information that must be provided for each relevant component of the system. In particular, for the devices interfacing with the robot, the robot positions must be defined beforehand in a device-attached coordinate system (CS) by the vendor. This CS must be detectable by the vision system of the robot by means of optical markers placed on the front side of the device. With that, the robot is capable of tending the machine by performing the pick-and-place type transportation of standard sample carriers. This basic use case is the primary scope of the LAPP-DT framework. The hardware scope is limited to simple benchtop and mobile manipulators with parallel grippers at this stage. This paper first provides an overview of relevant literature and state-of-the-art solutions, after which it outlines the framework on the conceptual level, followed by the specification of the relevant DT parameters for the robot, for the devices and for the facility. Finally, appropriate technologies and strategies are identified for the implementation.

Laboratories for the future

Research laboratories are gaining attention in the global sustainability rhetoric, as many overseas institutes attempt to improve the historically consumptive practices of research. Life science laboratories are estimated to contribute up to 2% of annual global plastic waste, and compared to other academic spaces, laboratories consume as much as 3 to 6 times more energy. Addressing laboratory sustainability is essential in the University of Otago achieving its Net Carbon Zero goal more readily. This research has investigated key factors of laboratory sustainability at the University of Otago, utilizing an observational case-study laboratory and survey to assess current research sustainability standards. The research laboratory case-study in the University of Otago department of Biochemistry shows keen engagement with sustainability principles in areas such as sustainable purchasing practices, resource management and cold storage, while areas such as energy usage exhibit room for improvement. A voluntary survey completed by members of the Biochemistry department indicated that researchers believed changes to waste reduction & recycling, community engagement & understanding, and purchasing practices would be the most impactful in improving laboratory sustainability. Implementing strategies such as purchasing takeback schemes, promoting participation in laboratory sustainability initiatives, and reducing consumption of disposable resources will be paramount in improving laboratory sustainability. Supervised by: Ray O'Brien, Sustainability Office, University of OtagoScholarship funded by: Waste Management NZ

Life sciences discovery and technology highlights

Automation for Life Science Laboratories. The automation of processes in all areas of the life sciences will continue to increase in the coming years due to an ever increasing number of samples to be processed, an increasing need to protect laboratory personnel from infectious material and increasing cost pressure. Depending on the requirements of the respective application, different concepts for automation systems are available, which have a different degree of automation with regard to data handling, transportation tasks, and the processing of the samples.

Accumulation of Heavy Metals by Some Aquatic Macrophytes in Two Streams along the Tiko-Douala Highway, Cameroon

Aims: This study was aimed at determining the ecological health of two roadside streams across the Tiko-Douala highway, Cameroon, and investigating the phytoremediation potentials of dominant aquatic macrophytes within these streams.
 Study Design: Water, sediments, and aquatic plant samples were collected from Camp 7 and Moqouo streams, and analyzed for ammonium nitrogen, nitrate nitrogen, phosphates, magnesium, cadmium, and lead, including other relevant water quality parameters.
 Place and Duration of Study: In the Life Science Laboratory of the University of Buea, and Soil and Water Laboratory of the University of Dschang, Cameroon between June 2021 and June 2022.
 Methodology: Six water samples and six sediment samples were collected per stream, and two of the most dominant plant species (Pennisetum purpureum Schumach (Poaceae) and Dicanthelium clandestinum L. (Poaceae) from Camp 7, Commelina benghalensis L. (Commelinaceae) and Oldenlandia corymbosa L.(Rubiaceae) from Moqouo stream) were collected for heavy metal analysis. Heavy metal concentration and bioaccumulation factors were determined for each plant.
 
 Results: High concentrations of phosphates were recorded in Camp 7 (1.19 mg/L) and Moqouo (2.17 mg/L) streams. Dissolved oxygen levels were 0.69 mg/L and 0.80 mg/L in Camp 7 and Moqouo respectively. Sediment cadmium levels ranged from 0.33± 0.02 to 0.63± 0.16 mg/kg, and lead ranged from 0.34 ±0.03 to 0.37 ± 0.12 mg/kg in Moqouo and Camp 7 respectively. 
 Conclusion: The Mouqouo and Camp 7 streams are rich in phosphates and thus not suitable for drinking and irrigation. Cadmium and lead in stream water were within the permissible limits but sediments contained higher concentrations than water and therefore they could be released gradually into the water column. All plants in the study except (Dicanthelium cladestinum) accumulated cadmium and lead, and could be potential plants for the phytoremediation of these metals.

Abstract 1931: Innovating Life Sciences Laboratory Training: Molecular Biology Laboratory Education Modules (MBLEMs) as a Model for Advanced Training at Diverse Institutions

Abstract 1931: Innovating Life Sciences Laboratory Training: Molecular Biology Laboratory Education Modules (MBLEMs) as a Model for Advanced Training at Diverse Institutions

Towards End-to-End Automated Microscopy Control using Holotomography: Workflow Design and Data Management

Microscopy has been a key tool involved in many discoveries in the life sciences over the past centuries. In the last 30 years in particular, enormous progress has been made in developing this measurement technique further to make researchers working with it more effective. To combine gains in reproducibility and efficiency resulting from these advancements in different research areas, we present for the first time a unified and comprehensive concept for an end-to-end automated microscopy workflow. To this end, we employ both robotic and computational methods as well as holotomography microscopy. Considering the physical preparation and cleanup of a measurement, the image acquisition, and the management and analysis of the resulting data, we give a fine-grained workflow description. We present the robotic system to perform the manual process steps and a Python package to standardize the resulting proprietary image (meta)data. For the other tasks, we identify suitable open-source tools to execute them and apply them to our setup. The choice of holotomography as a suitable microscopy technique to realize this workflow is elucidated. We envision that the adoption of an automated workflow paves the way toward a future life science laboratory where microscopy-based research is carried out more efficiently and reproducibly than in the past.

The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy

Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to new single-photon (SP) array detectors, which give access to an entirely new set of single-photon information. Together with the blooming of new SP LSM techniques and the development of tailored SP array detectors, there is a growing need for (i) DAQ systems capable of handling the high-throughput and high-resolution photon information generated by these detectors, and (ii) incorporating these DAQ protocols in existing fluorescence LSMs. We developed an open-source, low-cost, multi-channel time-tagging module (TTM) based on a field-programmable gate array that can tag in parallel multiple single-photon events, with 30 ps precision, and multiple synchronisation events, with 4 ns precision. We use the TTM to demonstrate live-cell super-resolved fluorescence lifetime image scanning microscopy and fluorescence lifetime fluctuation spectroscopy. We expect that our BrightEyes-TTM will support the microscopy community in spreading SP-LSM in many life science laboratories.

Facile methods for reusing laboratory plastic in developmental biology experiments

Plastic pollution negatively affects ecosystems and human health globally, with single-use plastic representing the majority of marine litter in some areas. Life science laboratories prefer pristine conditions for experimental reliability and therefore make use of factory standardized single-use plastic products. This contributes to overall plastic waste in the United States and globally. Here, we investigate the potential of reusing plastic culture dishes and subsequently propose methods to mitigate single-use plastic waste in developmental biology research laboratories. We tested the efficacy of bleach and ethyl alcohol in sterilizing used dishes. We then tested the feasibility of washing and reusing plastic to culture Xenopus laevis embryos subjected to various manipulations. Cleaning and reusing laboratory plastic did not affect the development or survival of X. laevis, indicating that these cleaning methods do not adversely affect experimental outcome and can be used to sterilize plastic before reuse or recycling. Lastly, we performed a survey of various life science laboratories to estimate both waste reduction and savings associated with recycling single-use plastics. Standardization of these procedures would allow research laboratories to benefit economically while practicing environmentally conscious consumption.

Evaluating a Novel Gas Sensor for Ambient Monitoring in Automated Life Science Laboratories.

Air pollution and leakages of hazardous and toxic gases and chemicals are among the dangers that frequently occur at automated chemical and life science laboratories. This type of accident needs to be processed as soon as possible to avoid the harmful side effects that can happen when a human is exposed. Nitrogen oxides and volatile organic compounds are among the most prominent indoor air pollutants, which greatly affect the lifestyles in these places. In this study, a commercial MOX gas sensor, SGP41, was embedded in an IoT environmental sensor node for hazardous gas detection and alarm. The sensor can detect several parameters, including nitrogen oxide index (NOx-Index) and volatile organic compound index (VOC-Index). Several tests were conducted to detect the leakage of nitrogen oxides and volatile organic compounds in different concentrations and volumes, as well as from different leakage distances, to measure the effect of these factors on the response speed and recovery time of the sensors used. These factors were also compared between the different sensors built into the sensor node to give a comprehensive picture of the system used. The system testing results revealed that the SGP41 sensor is capable of implementing the design purposes for the target parameters, can detect a small NO2 gas leakage starting from 0.3% volume, and can detect all the tested VOC solvents ≥ 100 µL.

Effective Cell Transfection in An Ultrasonically Levitated Droplet for Sustainable Technology.

The levitation methodology, which enables us to operate a contactless reaction without a container, is likely to be a revolutionary technology in the fields of chemistry and biology to reduce the plastic waste in life science laboratories. Here, the authors show that plasmid DNA can be effectively transfected into animal cells in a floating droplet of culture medium levitated using ultrasonic standing waves. The data indicate that there is no significant damage to the plasmid and cells during the levitating transfection time, and the transgene expression efficiency and cellular uptake in the droplet are significantly higher than those in the conventional tube, with and without shaking. These results suggest the consolidation of the endocytic uptake pathway into macropinocytosis, indicating that ultrasonic levitation induced a change in cell characteristics. This study suggests that transfection methodology using ultrasonic levitation has the potential to advance the current experimental procedures in the field of cell engineering, in addition to presenting a revolutionary containerless reactor for sustainable technology.