A microfluidic device for real-time monitoring of Bacillus subtilis bacterial spores during germination based on non-specific physicochemical interactions on the nanoscale level

Germination of spores into actively growing cells is a process essential for survival and pathogenesis of many microbes. Molecular mechanisms governing germination, however, are poorly understood in part because few tools exist for evaluating and interrogating the process. Here, we introduce an assay that leverages developments in microfluidic technology and image processing to quantitatively measure germination with unprecedented resolution, assessing both individual cells and the population as a whole. Using spores from Cryptococcus neoformans, a leading cause of fatal fungal disease in humans, we developed a platform to evaluate spores as they undergo morphological changes during differentiation into vegetatively growing yeast. The assay uses pipet-accessible microdevices that can be arrayed for efficient testing of diverse microenvironmental variables, including temperature and nutrients. We discovered that temperature influences germination rate, a carbon source alone is sufficient to induce germination, and the addition of a nitrogen source sustains it. Using this information, we optimized the assay for use with fungal growth inhibitors to pinpoint stages of germination inhibition. Unexpectedly, the clinical antifungal drugs amphotericin B and fluconazole did not significantly alter the process or timing of the transition from spore to yeast, indicating that vegetative growth and germination are distinct processes in C. neoformans. Finally, we used the high temporal resolution of the assay to determine the precise defect in a slow-germination mutant. Combining advances in microfluidics with a robust fungal molecular genetic system allowed us to identify and alter key temporal, morphological, and molecular events that occur during fungal germination.

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“…Some rely on changes in electrical conductivity of media 27 or polyaniline mesh 28 to track germination of Bacillus spp. spores.…”

Section: Discussionmentioning

confidence: 99%

Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination

et al. 2016

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“…Based on the presented results, a micro fluidic system (Figure 4C), analogous to that used previously [31,32], was designed for future testing of practical applications (Appendix B).…”

Section: Methodsmentioning

confidence: 99%

Micro- and Nanostructured Polyaniline for Instant Identification of Metal Ions in Solution

et al. 2019

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The unique properties of nanomaterials enable the creation new analytical devices. Polyaniline (PANI) micro- and nanofiber network, freestanding in the gap between two gold microelectrodes, has been used in a new nanodetector for metal ions in solutions. The gold electrodes were modified with the aid of alkanethiols, forming a self-assembled monolayer (SAM), which is able to block the ion current flow, but also to interact with metal ions when specific functional molecules are incorporated into the layer. The electric field of the trapped metal ions induces change of the electrical conductivity of polyaniline nanofibers in vicinity. A small injected sample (75 μL) of a solution of salt (about 0.5 μg of salt) was enough to induce a reproducible change in the electrical conductivity of polyaniline nano-network, which was registered as a function of time within 10–20 s. The response was proportional to the concentration of ions. It also depends on properties of ions, e.g., the ionic radius, which allows for identification of metal ions by analyzing the parameters of the signal: the retention time (RT), half width (HW), amplitude (A) and integral intensity (INT). The advantage of the new device is the instant responsiveness and easy operation, but also the simple construction based on organic (polymer) technology. The system is “open”—when learned and calibrated adequately, other metal ions can be analyzed. The nanodetector can be used in cases where monitoring of the presence and concentration of metal ions is important.

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“…This device is based on the changes in electrical conductivity of a polyaniline nanofiber network. Electrical changes are induced by the spores passing through a measuring interface and the signal depends on the state of the cell (dormant spores, germinated spores or vegetative cells) (Zabrocka, Langer, Michalski, Kocik, & Langer, 2015).…”

Section: Molecular Methods (Fig 2e-f-g)mentioning

confidence: 99%

Investigating germination and outgrowth of bacterial spores at several scales

Trunet

Carlin

Coroller

2017

Trends in Food Science & Technology

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Background: Spore-forming bacteria are a major cause of food spoilage and food poisoning. Spores thatresist physical and chemical treatments used in the food industry may germinate and multiply. Spore germination, outgrowth and growth constitute a complex and highly heterogeneous process.Scope and approach: Various techniques and methods can be used to observe the germination, outgrowth and early multiplication process of spore-forming bacteria and/or to quantify the impact of environmental conditions on its progress over time within a spore population. These techniques can be classified by different criteria: (i) the scale of analysis, from populations or cells to molecules, and (ii) the number of analyzed objects (cells) and (iii) the potential of the method to describe and/or quantify the impact of lethal or sub-lethal treatments or environmental conditions. Such treatments are applied to a spore population or a single spore and take into account parameters at the cellular level (growth capacity, morphological properties) to molecular level (proteomics, transcriptomics, spore molecularcomposition).Key findings and conclusion: A better understanding and quantification of the germination, outgrowth and growth process require the implementation of several complementary methods. Methods providing information at single and population levels, as well as at molecular and cellular levels, are essential to assess and control the fate of spore-forming bacteria development in food systems

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A microfluidic device for real-time monitoring of Bacillus subtilis bacterial spores during germination based on non-specific physicochemical interactions on the nanoscale level

Cited by 8 publications

References 16 publications

Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination

Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination

Micro- and Nanostructured Polyaniline for Instant Identification of Metal Ions in Solution

Investigating germination and outgrowth of bacterial spores at several scales

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