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Comparison of DPM-and PCM-derived parameters to be used for discrimination between B. licheniformis spores at different state
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One of the challenging tasks in monitoring studies is to estimate heterogeneity of microbial populations by the physiological state and potential viability of individual cells, especially with regard of their ability to withstand various environmental assaults. Previously, we described some approaches based on electron microscopy methods to discrim...
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p> Background : Environmental conditions in edges of vegetation fragments affect life cycle characteristics of the native biota (edge effect). We evaluated the edge effect on two Bursera species, which are representative of the mature community of the tropical deciduous forest (TDF) in Mexico.
Hypothesis . We expected a population structure reflec...
In the forest canopy, seeds of epiphytic plants encounter heterogeneous environments created by a combination of factors such as solar radiation, humidity, and host characteristics. Germination requirements may explain the species distribution in the canopy; however, more knowledge is essential. Germination of Rhipsalis baccifera, a widespread trop...
Citations
... Spores remain phase-bright. Germination can be monitored by phase contrast microscopy on individual spores or on limited spore populations (Pandey et al., 2013;Tychinsky et al., 2007); by Raman spectroscopy (Chen et al., 2006) to follow CaDPA release into culture medium (Zhang et al., 2012). Spores turn from phase-bright to phase dark. ...
... Both methods are based on the direct observation of cells, which may be marked with detectable probes. Phase contrast microscopy differentiates dormant spores (phase-bright spores) from germinated spores (phase-dark) by their higher refractive index and from vegetative cells by their smaller size (Chen, Huang, & Li, 2006;Hornstra, Ter Beek, Smelt, Kallemeijn, & Brul, 2009;Tychinsky et al., 2007). Various physical media may be used for microscopic observation of spore germination and outgrowth. ...
... This technique has shown some potential to study the activation of spores by heat, a sublethal treatment promoting the germination of many species of spore-forming bacteria. Dormant spores (refractive) and germinated spores of B. licheniformis had significantly different phase thickness values (Tichinsky, Kretushev, & Luskinovich, 2006;Tychinsky et al., 2007). Moreover, heat inactivated spores had a lower phase thickness value than dormant spores. ...
Background Spore-forming bacteria are a major cause of food spoilage and food poisoning. Spores that resist 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 molecular composition). 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.
The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this article, we consider the applications of the CPM method to imaging different cells and energy-transducing intracellular organelles (mitochondria and chloroplasts). Experimental data presented below demonstrate that the optical path length difference of the object, which is the basic optical parameter measured by the CPM method, can serve as an indicator of metabolic states of different biological objects at cellular and subcellular levels of structural organization.
Initially, it has been shown that the phase thickness and refractivity (the latter interpreted as the difference of the refractivity indices of an object and surrounding medium) depend on the functional state of mitochondria. The refractivity of various objects decreased in response to energy depletion. This dependence was then demonstrated for other biological objects such as cyanobacteria, chloroplasts and human cells. This general response brought about the hypothesis of a certain "universal" factor that links the variable (or metabolic) component of refractivity with the object's functional state. However, the origin of this phenomenon remains unknown. Our hypothesis is founded on the dependence of polarization of bound water molecules and the activity of metabolic processes. Here, we show the results of measurements of metabolic component of refractivity different bio-objects (mitochondria, chloroplasts, spores, cancer cells) obtained using the Coherent Phase Microscope "Airyscan". Estimations indicated high (up to n approximately = 1.41-1.45) values for the equivalent refractive index of structured water in cells.
We develop a method of coherent phase microscopy (CPM) for direct visualization of nonfixed, nonstained mammalian cells (both cultured cells and freshly isolated tumor biopsies) followed by computer-assisted data analysis. The major purpose of CPM is to evaluate the refractive properties of optically dense intracellular structures such as the nucleus and the nucleoli. In particular, we focus on quantitative real-time analysis of the nucleolar dynamics using phase thickness as an equivalent of optical path difference for optically nonhomogenous biological objects. Pharmacological inhibition of gene transcription leads to a dramatic decrease of the phase thickness of the nucleoli within the initial minutes of cell exposure. Furthermore, the acute depletion of intracellular ATP pool, depolymerization of microtubules and inhibition of DNA replication resulted in a rapid decrease of the nucleolar phase thickness. These optical effects were paralleled by segregation of nucleolar components as documented by electron microscopy. Thus, CPM detects early changes of nucleolar dynamics, in particular, the nucleolar segregation as part of general cellular response to cytotoxic stress, regardless of whether the nucleolus is or is not the primary target of the toxin. CPM is applicable for monitoring and quantitative analysis of the "nucleolar stress" in living mammalian cells.
