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Scientific Reports Dec 2022In nature, bacteria prevailingly reside in the form of biofilms. These elaborately organized surface-bound assemblages of bacterial cells show numerous features of...
In nature, bacteria prevailingly reside in the form of biofilms. These elaborately organized surface-bound assemblages of bacterial cells show numerous features of multicellular organization. We recently showed that biofilm growth is a true developmental process, which resembles developmental processes in multicellular eukaryotes. To study the biofilm growth in a fashion of eukaryotic ontogeny, it is essential to define dynamics and critical transitional phases of this process. The first step in this endeavor is to record the gross morphological changes of biofilm ontogeny under standardized conditions. This visual information is instrumental in guiding the sampling strategy for the later omics analyses of biofilm ontogeny. However, none of the currently available visualizations methods is specifically tailored for recording gross morphology across the whole biofilm development. To address this void, here we present an affordable Arduino-based approach for time-lapse visualization of complete biofilm ontogeny using bright field stereomicroscopy with episcopic illumination. The major challenge in recording biofilm development on the air-solid interphase is water condensation, which compromises filming directly through the lid of a Petri dish. To overcome these trade-offs, we developed an Arduino microcontroller setup which synchronizes a robotic arm, responsible for opening and closing the Petri dish lid, with the activity of a stereomicroscope-mounted camera and lighting conditions. We placed this setup into a microbiological incubator that maintains temperature and humidity during the biofilm growth. As a proof-of-principle, we recorded biofilm development of five Bacillus subtilis strains that show different morphological and developmental dynamics.
Topics: Time-Lapse Imaging; Microscopy; Bacteria
PubMed: 36476631
DOI: 10.1038/s41598-022-24431-y -
Environmental Entomology Feb 2023Beauveria bassiana (Balsamo) Vuillemin infects a wide variety of insects, including the green peach aphid, Myzus persicae (Sulzer). Volatiles emitted from B. bassiana...
Beauveria bassiana (Balsamo) Vuillemin infects a wide variety of insects, including the green peach aphid, Myzus persicae (Sulzer). Volatiles emitted from B. bassiana can act as semiochemical attractants or repellents, with most responses reported to date resulting in insects avoiding B. bassiana. Since insects can detect 'enemy-specific volatile compounds', we hypothesized the preference behavior of M. persicae would be influenced by volatile emissions from B. bassiana. We conducted Petri dish and Y-tube olfactometer bioassays to characterize the preference of M. persicae to B. bassiana strain GHA. During Petri dish bioassays, more apterous and alate M. persicae were recorded in the vicinity of agar colonized by B. bassiana compared to agar, or Fusarium proliferatum (Matsushima) Nirenberg and Ambrosiella grosmanniae Mayers, McNew, & Harrington as representatives of nonentomopathogenic fungi. Petri dish bioassays also determined that apterous and alate M. persicae preferred filter paper saturated with 1 × 107, 1 × 106, and 1 × 105B. bassiana conidia/ml compared to Tween 80. Y-tube bioassays documented that more apterous and alate M. persicae oriented upwind to volatiles from B. bassiana mycelia compared to agar. Apterous and alate Myzus persicae were also preferentially attracted to 1 × 107 and 1 × 106B. bassiana conidia/ml compared to Tween-80 during Y-tube bioassays. These results complement a previous finding that the mosquito Anopheles stephensi (Diptera: Culicidae) Liston is attracted to volatiles from B. bassiana. Future studies aimed at characterizing the olfactory mechanism leading to the attraction of M. persicae to B. bassiana could aid in optimizing lure-and-kill strategies.
Topics: Animals; Beauveria; Aphids; Agar; Spores, Fungal; Pest Control, Biological
PubMed: 36421055
DOI: 10.1093/ee/nvac100 -
History and Philosophy of the Life... Nov 2017Microbial diversity has become a leitmotiv of contemporary microbiology, as epitomized in the concept of the microbiome, with significant consequences for the...
Microbial diversity has become a leitmotiv of contemporary microbiology, as epitomized in the concept of the microbiome, with significant consequences for the classification of microbes. In this paper, I contrast microbiology's current diversity ideal with its influential predecessor in the twentieth century, that of purity, as epitomized in Robert Koch's bacteriological culture methods. Purity and diversity, the two polar opposites with regard to making sense of the microbial world, have been operationalized in microbiological practice by tools such as the "clean" Petri dish versus the "dirty" Winogradsky column, the latter a container that mimics, in the laboratory, the natural environment that teems with diverse microbial life. By tracing the impact of the practices and concepts of purity and diversity on microbial classification through a history of techniques, tools, and manuals, I show the shifts in these concepts over the last century. Juxtaposing the dominant purity ideal with the more restricted, but continuously articulated, diversity ideal in microbial ecology not only provides a fresh perspective on microbial classification that goes beyond its intellectual history, but also contextualizes the present focus on diversity. By covering the period of a century, this paper outlines a revised longue durée historiography that takes its inspiration from artifacts, such as Petri dish and the Winogradsky column, and thereby simple, but influential technologies that often remain invisible. This enables the problem of historical continuity in modern science to be addressed and the accelerationist narratives of its development to be countered.
Topics: Historiography; History, 19th Century; History, 20th Century; Microbiological Techniques; Microbiology
PubMed: 29188459
DOI: 10.1007/s40656-017-0175-9 -
Materials (Basel, Switzerland) Aug 2019The wound-healing assay is commonly and widely used for investigating collective cell migration under various physical and chemical stimuli. Substrate-coating materials...
The wound-healing assay is commonly and widely used for investigating collective cell migration under various physical and chemical stimuli. Substrate-coating materials are shown to affect the wound-healing process in a cell-type dependent manner. However, experiment-to-experiment variations make it difficult to compare results from different assays. In this paper, a modified barrier wound-healing assay was reported for studying the wound-healing process on different substrates in one single petri dish. In short, half of a dish was covered with the tape, and coating materials, poly-l-lysine and gelatin, were applied to the surface. After peeling off the tape, half of the surface was coated with the desired material. Then a customized barrier was placed inside the dish to create the wound. The results indicated that surface coating did not affect cell proliferation/viability, and the wound-healing rate increased in coated surfaces compared to uncoated ones. The present study provides a platform for further understanding the mechanisms of substrate coating-dependent wound-healing processes.
PubMed: 31470524
DOI: 10.3390/ma12172775 -
SLAS Technology Apr 2023The spot assay of the budding yeast Saccharomyces cerevisiae is an experimental method that is used to evaluate the effect of genotypes, medium conditions, and...
The spot assay of the budding yeast Saccharomyces cerevisiae is an experimental method that is used to evaluate the effect of genotypes, medium conditions, and environmental stresses on cell growth and survival. Automation of the spot assay experiments from preparing a dilution series to spotting to observing spots continuously has been implemented based on large laboratory automation devices and robots, especially for high-throughput functional screening assays. However, there has yet to be an affordable solution for the automated spot assays suited to researchers in average laboratories and with high customizability for end-users. To make reproducible spot assay experiments widely available, we have automated the plate-based yeast spot assay of budding yeast using Opentrons OT-2 (OT-2), an affordable liquid-handling robot, and a flatbed scanner. We prepared a 3D-printed mount for the Petri dish to allow for precise placement of the Petri dish inside the OT-2. To account for the uneven height of the agar plates, which were made by human hands, we devised a method to adjust the z-position of the pipette tips based on the weight of each agar plate. During the incubation of the agar plates, a flatbed scanner was used to automatically take images of the agar plates over time, allowing researchers to quantify and compare the cell density within the spots at optimal time points a posteriori. Furthermore, the accuracy of the newly developed automated spot assay was verified by performing spot assays with human experimenters and the OT-2 and quantifying the yeast-grown area of the spots. This study will contribute to the introduction of automated spot assays and the automated acquisition of growth processes in conventional laboratories that are not adapted for high-throughput laboratory automation.
Topics: Humans; Saccharomyces cerevisiae; Agar; Robotics; Automation; Genotype
PubMed: 36503082
DOI: 10.1016/j.slast.2022.12.001 -
Protein Science : a Publication of the... Apr 2016From humble beginnings of a contaminated petri dish, β-lactam antibiotics have distinguished themselves among some of the most powerful drugs in human history. The... (Review)
Review
From humble beginnings of a contaminated petri dish, β-lactam antibiotics have distinguished themselves among some of the most powerful drugs in human history. The devastating effects of antibiotic resistance have nevertheless led to an "arms race" with disquieting prospects. The emergence of multidrug resistant bacteria threatens an ever-dwindling antibiotic arsenal, calling for new discovery, rediscovery, and innovation in β-lactam research. Here the current state of β-lactam antibiotics from a structural perspective was reviewed.
Topics: Anti-Bacterial Agents; Humans; Structure-Activity Relationship; beta-Lactam Resistance; beta-Lactamase Inhibitors
PubMed: 26813250
DOI: 10.1002/pro.2889 -
Journal of Visualized Experiments : JoVE Feb 2020Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of...
Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation. The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins β-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed "domes". The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce β-casein and WAP. Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated Rac increase differentiation, high Rac levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of Rac.
Topics: Animals; Cadherins; Cell Culture Techniques; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Culture Media; Epithelial Cells; Female; Mammary Glands, Animal; Mice; Milk Proteins; Spheroids, Cellular; rac GTP-Binding Proteins
PubMed: 32176212
DOI: 10.3791/60147 -
Investigative Ophthalmology & Visual... Nov 2017To quantify cell survival and tissue structure preservation of porcine cornea stored in a bioreactor (BR) that recreates a transcorneal pressure gradient equivalent to...
PURPOSE
To quantify cell survival and tissue structure preservation of porcine cornea stored in a bioreactor (BR) that recreates a transcorneal pressure gradient equivalent to intraocular pressure (IOP) and renews the medium.
METHODS
A BR comprising endothelial and epithelial chambers was machined in a biocompatible material. The porcine cornea, securely held, separated the chambers. Medium flow and pressure inside the endothelial chamber were maintained by a peristaltic pump. In the epithelial chamber, the corneal surface was alternatively exposed to air and a specific medium. Two transparent windows allowed thickness measurement by optical coherence tomography without opening the BR. Porcine corneas stored in the BR-on (pressure 20 mm Hg, flow 5 μL/min, temperature 31°C) were compared with (1) BR-off (no pressure or flow); (2) organ culture; and (3) Petri dish with agar on the endothelial side. Epithelial and limbal structure and differentiation, corneal transparency and thickness, and endothelial viability were compared after 7 days of storage and with fresh corneas.
RESULTS
Corneas stored in the BR-on were thinner and more transparent than those stored with the other methods. The BR-on preserved a stratified and differentiated (K3/K12+) corneal epithelium and undifferentiated basal limbal cells with stemness markers (K3/K12-; ABCB5, K14, p75+), as well as endothelial integrity.
CONCLUSIONS
By recreating equivalent IOP and medium renewal, the BR obtained unprecedented storage quality of porcine corneas and preserved their main epithelial, limbal, and endothelial characteristics.
Topics: Animals; Bioreactors; Cell Count; Cell Differentiation; Cell Survival; Cornea; Equipment Design; Models, Animal; Organ Culture Techniques; Organ Preservation; Swine
PubMed: 29164231
DOI: 10.1167/iovs.17-22218 -
Journal of Neuroscience Methods Jan 2018Microscope chambers that accept glass coverslips with cultured cells are often used to monitor intracellular Ca concentration ([Ca]) during cell superfusion....
BACKGROUND
Microscope chambers that accept glass coverslips with cultured cells are often used to monitor intracellular Ca concentration ([Ca]) during cell superfusion. Unfortunately, the experimental maneuvers associated with the coverslip installation in these chambers (medium removal and re-application) trigger unintended [Ca] elevations.
NEW METHOD
To prevent these [Ca] elevations, a Petri dish insert has been constructed. The insert features a superfusion-optimized well to grow cell cultures. After this insert is removed from the Petri dish, the well retains the medium. This feature allows the inserts to be installed in microscope chambers while keeping the cells submerged at all times.
RESULTS
These inserts were used to test the impact of a transient medium removal from the well (an equivalent of a coverslip removal from the medium) on [Ca] in primary murine cortical neurons and astrocytes, and in HEK-293 cells. In all of these models, the medium removal/re-application caused a micromolar [Ca] spike. While in neurons this spike was caused by a Ca influx, in astrocytes and HEK-293 cells, it was caused by a Ca release from intracellular stores. After the spike, a subpopulation of neurons failed to restore low [Ca]; in 24% of the astrocytes, the spike triggered [Ca] oscillations. However, prior to the spike, [Ca] was low and uniform in all these cells.
COMPARISON WITH EXISTING METHOD(S)
The new method avoids the artificially-induced [Ca] elevations that take place during the handling of glass coverslips with cultured cells.
CONCLUSIONS
The new method allows monitoring [Ca] without disturbing the basal [Ca] levels.
Topics: Animals; Astrocytes; Calcium Signaling; Cell Culture Techniques; Coculture Techniques; Culture Media; Female; HEK293 Cells; Humans; Male; Mice, Inbred C57BL; Neurons; Optical Imaging; Primary Cell Culture
PubMed: 29132810
DOI: 10.1016/j.jneumeth.2017.11.007 -
Sensors (Basel, Switzerland) Oct 2023Colony-Forming Unit (CFU) counting is a complex problem without a universal solution in biomedical and food safety domains. A multitude of sophisticated heuristics and...
Colony-Forming Unit (CFU) counting is a complex problem without a universal solution in biomedical and food safety domains. A multitude of sophisticated heuristics and segmentation-driven approaches have been proposed by researchers. However, U-Net remains the most frequently cited and used deep learning method in these domains. The latter approach provides a segmentation output map and requires an additional counting procedure to calculate unique segmented regions and detect microbial colonies. However, due to pixel-based targets, it tends to generate irrelevant artifacts or errant pixels, leading to inaccurate and mixed post-processing results. In response to these challenges, this paper proposes a novel hybrid counting approach, incorporating a multi-loss U-Net reformulation and a post-processing Petri dish localization algorithm. Firstly, a unique innovation lies in the multi-loss U-Net reformulation. An additional loss term is introduced in the bottleneck U-Net layer, focusing on the delivery of an auxiliary signal that indicates where to look for distinct CFUs. Secondly, the novel localization algorithm automatically incorporates an agar plate and its bezel into the CFU counting techniques. Finally, the proposition is further enhanced by the integration of a fully automated solution, which comprises a specially designed uniform Petri dish illumination system and a counting web application. The latter application directly receives images from the camera, processes them, and sends the segmentation results to the user. This feature provides an opportunity to correct the CFU counts, offering a feedback loop that contributes to the continued development of the deep learning model. Through extensive experimentation, the authors of this paper have found that all probed multi-loss U-Net architectures incorporated into the proposed hybrid approach consistently outperformed their single-loss counterparts, as well as other comparable models such as self-normalized density maps and YOLOv6, by at least 1% to 3% in mean absolute and symmetric mean absolute percentage errors. Further significant improvements were also reported through the means of the novel localization algorithm. This reaffirms the effectiveness of the proposed hybrid solution in addressing contemporary challenges of precise in vitro CFU counting.
PubMed: 37837169
DOI: 10.3390/s23198337