saccharomyces cerevisiae under microscope 400xchemical that dissolves human feces in pit toilet
batchculturesubstrate unlimited batch growth of a culture at quasi-steady-state conditions with max in constant volume in minimal medium with glucose as a sole carbon and energy source, granularitythe relative arbitrary value (measured by side scatter (SSC) laser light) used in flow cytometry to index an intracellular morphological complexity (i.e. The phases of the cell cycle are separated by molecular control mechanisms (e.g. The study of budding of Saccharomyces with the However, it may be present in semihard and hard cheese including Cheddar cheese. The anaerobic batch growth was performed in Aquatron (INFORS HT, Switzerland) orbital water bath shaker (250 rpm) with gas-lid under constant nitrogen flow (0.5 L/h) through the shaker (pO2=0%). Figure 10.2. The significance of species such as S. cerevisiae as spoilage organisms in cheeses is not well understood and it has been suggested that rather than causing spoilage, it may play a role in flavour development during the maturation of cheeses. Search for other works by this author on: Cell volume is an important parameter for mathematical modeling of the metabolic cellular processes (Reich and Selkov, \begin{equation} For the screens described here, the promoter for the pheromone-responsive gene FUS1 (designated FUS1p) was ligated to HIS3, a gene encoding imidazoleglycerolphosphate dehydratase and required for histidine biosynthesis in yeast. 3). There are two major checkpoints in yeast cell cycle: (i) G1 or so-called Start and (ii) spindle assembly or so-called Finish (Chen etal.2004). 4K video of yeast (Saccharomyces cerevisiae) seen at 100x Quantitation of signals is shown at the bottom right. The nervous system of Hydra is a nerve net. They are small organisms, ranging from 340 micrometers in some of the approximately 1,500 species. budding (Vanoni, Vai and Frascotti 1984). The addition of carrier DNA also promotes the uptake of vector DNA. If to compare the same values of the biomass yields achieved in both temperature regions, then it is obvious that the cells from 3340C region have significantly lower granularity (Fig. macromolecular composition, content of organelles, content of various deposits, etc) of yeast cells, which obviously can be detected by FC as the varying intracellular morphological complexity or so-called intracellular granularity. The species also causes spoilage of carbonated soft drinks and fruit drinks, sports drinks, pured fruits and canned fruit products. There are several known criteria for passing each checkpoint. Contamination with yeasts may arise from the fruit, from insect vectors or from the processing environment. (B) RNase H/Northern blotting analysis of HSP104 3 ends as described in A except that oligo(dT) was omitted from the RNase H reactions. Mutant values were normalized to wild type from the same time point. Content may be subject to copyright. During wine fermentation, indigenous strains of S. cerevisiae may produce undesirable characteristics. Webstrains under various conditions. It is obvious that the population of cells, where cells are three-dimensional particles (Fig. At the same time, it is known that content of intracellular organelles also varies in dependence on the environmental factors. at temperatures 18.526.3C, the budding activity ( f2) is relatively high (Fig. To our knowledge, there is no systematic information on the variability of intracellular morphology in dependence on the growth temperature. Means for Classification: Saccharomyces cerevisiae is in the fungi kingdom. Consequently, as the reasonable compromise for the problem, the shape of the yeast cells has been approximated to the sphere with diameter i (Fig. Final biomass concentration achieved in anaerobic batch culture, was reported in Zakhartsev etal. 1. Dependence of averaged cell diameter of yeast Saccharomyces cerevisiae CEN.PK 1137D in substrate unlimited anaerobic batch growth on (A) growth temperature and (B) on maximum specific growth rate achieved at corresponding temperature ( max, reported in Zakhartsev etal.2015). Saccharomyces cerevisiae has been reported to form approximately 25% of the yeast population of fruit juice concentrates. The pellet was twice washed with 10 mL of ice-cold 0.9% NaCl solution and dried out at 115C overnight. 3). S. cerevisiae view at an optical microscope, 40 X increased. These mutants also fail to produce proteins from heat-inducible genes, for example, HSP104 (Jensen et al., 2004, unpublished observations). This approach yields similar results as the RNase H/Northern blotting approach (see for example, Fig. WebContent from this work may be used under the terms of the CreativeCommonsAttribution 3.0 licence. Averaged diameter of single mother cells in G1 growth phase (Fig. Additionally, the structure of the population of the exponentially growing batch culture also varies in dependence of the growth temperature (Fig. The peak with 1 is formed by the fraction of single cells in G1-growth phase in the population, whereas the peak with 2 is formed by the fraction of the budding cells in S/G2/M-growth phases in the population [10]. Saccharomyces cerevisiae, also known as baker's yeast, is a unicellular fungus that is used for the purpose of making bread and other Thus, the parameters of cell size distribution histogram are insufficient in order to calculate the semi-axes ( a, b, c) of yeast cells (Fig. This can be explained that in this temperature region, the larger fraction of glucose is metabolized to the end-products (e.g. Averaged diameters of the single and the budding cells of yeast Saccharomyces cerevisiae CEN.PK 1137D were measured under different isothermal growth conditions between 5 and 40C (Table 1). The most common variety youre likely to encounter is Saccharomyces cerevisiae, which is used for the edible products we mentioned earlier. 10.2C and D). The diameter of averaged single cells exponentially decays from 10.2 m at 5C down to asymptotic value at around 8 m Saccharomyces cerevisiae can exist in two different forms: haploid or diploid. Total-cell RNA samples were collected from cells after a 5- and a 30-min temperature shift to 37 C. Saccharomyces cerevisiae is less commonly associated with vegetables, but it has been isolated from spoiled, softened cucumbers in brine. While direct correlation was not yet achieved, the system already offers the possibility to verify the state of the identical population of cells by fluorescence microscopy immediately before freezing and processing for transmission electron microscopy. 556288; Cat.No.556286). S.cerevisiae under the microscope [12] - ResearchGate Saccharomyces cerevisiae (bakers yeast) can be employed by regulators of G-protein signaling (RGS) researchers for a variety of purposes. Quantitation is shown on the right. Yeast size and intracellular granularity were studied by flow cytometry (FACSVantage SE from Becton Dickinson). Within each isothermal growth conditions, the biomass increment was followed over the time and different growth parameters of the biomass (e.g. maximum specific growth rate, biomass yield, specific rate of glucose consumption) were additionally determined from the same cultures (Table1; as exemplified in Fig. Approximated surface area of an averaged cell in population (equation (5)). The cell suspension was not sonicated, since this results in partial cell disruption. The existence of two distinctive temperature regions (531C vs. 3340C) in cellular morphology becomes even more obvious when SSC-index is plotted against of the biomass yield on glucose (Fig. At G1-checkpoint, a cell can be arrested if DNA damage is detected, mating pheromone is present, presence of specific gene products, the lack of protein levels or the cell has not reached the critical cell size (i.e.VTV), then it is unable to undergo the Start transition which commits the cell to a new round of DNA synthesis and mitosis, e.g. There is a critical cell size/volume [|$V_{TV}^{critical}$|] that microbial cells must reach in order to initiate the cell division. 6). Microscope Nevertheless, the SSC-index is significantly getting lower in the temperature region 3340C where the cellular rate of maintenance is increased 12-folds (Zakhartsev etal.2015). Transformation techniques are similar to those applied for Escherichia coli, but are modified to account for differences in the cell wall complexity of yeast. There is superposition of two major factors that result in the normal Gaussian distribution of the cell sizes of the yeast cells in population measured by FC: (i) natural variability in geometric shapes (e.g. Fraction of budding cells (in S/G2/M phases with 2; defined at Fig. 1) within the cell population and duration of budding period ( tb; equation (7)) in dependence on (A) growth temperature and (B,C) maximum specific growth rate of the biomass ( max) in anaerobic glucose unlimited batch cultures of yeast Saccharomyces cerevisiae CEN.PK 1137D. The resulting larger complex reaches the cell surface more easily for subsequent uptake. This is confirmed by very high SSC-index (Fig. The wavelength of 660 nm is often used in common laboratory practice because there are no endogenous chromophores in microbes that absorb the light of this wavelength. Haploid Saccharomyces cerevisiae exists in two mating types, designated a and (see J. Kurjan32 and L. Bardwell et al.33 for general reviews of the yeast pheromone response pathway). After transcription induction, most HSP104 RNA is detectable in the cytoplasm of wild-type cells. Figure 10.3. FUNGI Red Mountain Microbiology - Maricopa budding phase; STARTG1-checkpoint; FINISHspindle assembly checkpoint; td doubling time of the biomass [ h], assuming exponential growth (equation (4)); td duration of the S/G2/M-phase, i.e. (B) HSP104 RNA FISH analysis of sub2201 cells heat treated for 15min at 42 C and fixed immediately (left) or left for 30min after transcription shut-off before fixation (right). (A) Schematic of the yeast pheromone response pathway. 1B.3) exclusively depends on the inner morphological complexity of a cell (i.e. Consequently, it is expected that density of the biomass packing should vary in dependence of the growth conditions. The budding cell is not feeding and the material growth of the bud occurs at the expenses of deposits, which are the only source of primary elements and energy in this period, i.e. Hydra tentacles captured at 100x under the microscope. These include plasmid size, DNA configuration and quality, host strain, and selection procedures. Anabaena 400X: General Biology Lab: Loyola University Chicago Unfortunately, the flow cytometer BD FACSVantage SE cannot measure the cell concentration in the collected samples, therefore it was not possible to calculate x. Additionally, the author would like to thank Prof.Peter Scheurich (Institute of Cell Biology and Immunology, University of Stuttgart, Germany) for the experimental support, Achim Hauck (IBVT, University of Stuttgart, Germany) and Dr.Xuelian Yang (Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University, Beijing, China) for the research assistance, Dr. Pavlo Holenya (Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Germany) for the discussion of the results. For example, if you are looking for what Stachybotrys chartarum spores and growth structures or conidiophores look like under the microscope, just scroll down to the "S" section of our identification photographs of mold under the microscope. The first section addresses expression of RGS proteins in yeast: how to chose an expression vector, how to transform the vector into yeast, and how to check for expression. Nevertheless, they are reported in Table1 since they are used in the data analysis in this research and the method of their calculation is briefly described in Supplement 2 (Supporting Information). In screening yeast for cDNAs that express G-protein pathway activators, the cell cycle arrest normally associated with pheromone pathway activation can be circumvented by deletion of FAR1,34 thereby uncoupling pathway activation from growth arrest. x, equation (3)) propagates due to cell growth in course of the G1-phase. Flame an inoculating loop and allow it to cool. Monitoring of an optical density of a cell suspension at 660 nm (OD660) is used in biotechnology as an express method to estimate the biomass content (Hulst 1957; Koch 1994). 1. Observing Yeast Under The Microscope Microscope Club This mutant arises spontaneously when a sequence of the DNA in the mitochondria becomes defective to form a flawed mitochondrial genome. temperature dependent passage through the checkpoints in the cell cycle, i.e. cell pigmentation, total DNA/RNA content, cell cycle analysis, cell kinetics, proliferation, chromosome analysis, detection of variously labeled biomarkers, etc]. Haziness results from the presence of wild, non-flocculating strains in beer. The flow cytometry allows accurate monitoring of the optical properties of individual cells from the population. It means that at low growth rates ( max<0.1 h 1) observed at temperatures <18.5C lesser amount of cells start budding, so they perhaps either (i) are arrested in the G1-checkpoint where they keep on growing until fulfilment of another passage-criterion or (ii) they exit from the cell cycle into G0-phase (Boender etal.2011). As it was shown previously (Zakhartsev etal.2015), there are two temperature regions (531C vs. 3340C) for yeast Saccharomyces cerevisiae CEN.PK 1137D where rglc differently depends on max, and observed difference is due to 12-folds increased maintenance rate in temperature region 3340C versus 531C. Yeast counts in products such as apple turnovers may reach up to 106cfuml1 resulting in fermentative spoilage and blown packages. (2015). As a sum: step-wise increase of maintenance rate (which has been observed in 3340C growth temperatures; Zakhartsev etal.2015) is accompanied by a relative increase of glucose consumption rate and also with a significant morphological shift in the intracellular structures, which potentially lead to the reduction of the biomass density. max) (Nissen etal.1997; Lange and Heijnen 2001). Saccharomyces cerevisiae - an overview | ScienceDirect Topics Growth experiments were run always in duplicate (two flasks). 10.2A). Hence, transcription site-associated RNAs most likely reflect the low level pool of stable HSP104 transcripts detectable in the sub2201 mutant strain (Fig. Reed B. Wickner, Rosa Esteban, in Advances in Virus Research, 2013. Thus, if the max reduction is accompanied by increasing fraction of the budding cells in the population then it may indicate cell arrest in the Finish checkpoint (for references see Fig. Where: G1 G1-growth phase; S/G2/MS/G2/M-growth phases, i.e. However, according to our knowledge, there is no systematic research on the investigation of cell size variability of yeast Saccharomyces cerevisiae under different temperature growth conditions. Saccharomyces cerevisiae is widespread in its occurrence in nature, on fruits, leaves and nectars, and although it is not commonly associated with spoilage of fresh fruits it is often implicated in the spoilage of processed fruit products. The carbohydrates mainly stored in form of glycogen granules in the yeasts, which makes the cytosol optically inhomogeneous. Make a wet mount of the culture (SMALL inoculum) in a drop of lactophenol cotton blue (10X and 40X). As expected, there is a linear relationship between them. Oxford University Press is a department of the University of Oxford. cerevisiae under However, employing of the fluorescent labels (both endogenous and exogenous being specifically attached to biomarkers) enormously expands the list of measured parameters [e.g. 1) (min/max). The maximal SSC value was observed at 5C, whereas the minimal value at 33C, and then again it increases towards 40C (Fig. Arbitrary units, was reported by flow cytometer. [12] Interaction between bioreceptors and analytes is called viability 3). Due to integrative nature of the OD660 value, it is impossible to relate observed step-wise shift to any specific contributors (e.g.N, , cell opacity). Because S. cerevisiae can tolerate ethanol concentrations of up to 15%, it may occasionally spoil alcoholic beverages, including wine and beer. 4B). The possible causes of the difference have been attributed to the acute increase in the maintenance rate in the supraoptimal temperature region (i.e. The two-peak size distribution histogram (exemplified at Fig. Saccharomyces cerevisiae CKII has been purified to homogeneity and characterized both structurally and functionally (17, 39; for review, see 16). 1B.2), where 1 < 2. Particularly, the fraction of the budding cells ( f2; fraction of cells with 2 denoted at Fig. WebSaccharomyces cerevesiae Yeast reproduce asexually by budding, small daughter cells arising from the mother cell. In S. cerevisiae, this can be achieved with several approaches, such as using repressible promoters, temperature-sensitive RNAPII alleles, or simply treating cells with transcription inhibitors, such as thiolutin (Caponigro and Parker, 1996). The introduction of an essential biosynthetic gene whose expression is driven by a pheromone-responsive promoter provides the means for identifying pheromone pathway activators through a growth-based screen. 7), therefore there should not be over-densification of the packing of the cytoplasmic content. Source publication Boerhaaves syndrome Hydra extend their body to maximum length when feeding and slowly extend their tentacles. Nuclear retention of HSP104 RNA in the sub2201 mutant. The relationship between averaged cell granularity (i.e. The observation that yeast cells accumulate trehalose when deprived of glucose, nitrogen, sulfur, or phosphorus suggests that reserve carbohydrate accumulation is a general response to various types of nutrient limitation (37). 1A). 10.3B; Rougemaille et al., 2007). 8), because the majority of cells in the population are the single cells which are arrested at G1-checkpoint (or may be some cells even can be in G0-phase (Boender etal.2011) at extremely low max), therefore they keep on growing until passage through the G1-checkpoint. amounts of ribosomes, mitochondria; Farewell and Neidhardt (1998)). The total approximated intracellular volume ( VTV) and approximated surface-to-volume ratio ( STS/VTV) of an averaged cell in population were calculated for the averaged yeast cell as the function of the growth temperature and the specific growth rate (Fig. This is possible by plunge-freezing of an optically transparent sample sandwich, so that the temporal resolution is only determined by the transfer speed from the fluorescence microscope to the freezing device. Put the sample side facing up and Datasets at Figs 4 and 5 were fit to one-phase exponential decay function to reveal the asymptotes. Therefore, it is expected that temperature induced variation in max must be reflected in variability of the intracellular content (i.e. (2015); hereby, we would like to add that the early observed metabolic adjustments achieved in course of the temperature dependent growth are accompanied by independently observed changes in the intracellular morphology, which are somehow related to the energy metabolism. The experiments and conceptual logic leading to this conclusion are discussed here, and detailed experimental protocols are provided at the end of the chapter. Dashed and shaded area is 95% Confidence Interval of one-phase exponential decay regression curve. Saccharomyces cerevisiae can synthesize and degrade trehalose and, depending on the environmental conditions and the stage of the life cycle, trehalose can represent less than l%, or more than 23%, of the dry weight of cells (37, 42, 43). For example, high content of ribosomes was observed in some microbes at low growth rates (Farewell and Neidhardt 1998). Alternatively, the FC is suitable technology that allows simultaneous multiparametric analysis of a cell suspension with or without employing fluorescent probes/labels. 1A). Be careful to avoid bubbles.4. Consequently, the population has a wide range of sizes and increased average approximated cellular volumes (Figs 4 and 5; Fig S1, Supporting Information). Growth temperature has the profound effect on the specific growth rate of the biomass of yeast (Zakhartsev etal.2015) through affecting the duration of the cell cycle (Vanoni, Vai and Frascotti 1984): the lower temperature, the slower is the cell cycle and therefore the longer doubling time of the biomass (equation (4), Fig. 4B) resembles conclusion derived in Porro etal. It is known that the increase in G1-phase duration is accompanied by a strong increase in the levels of the reserved carbohydrate. From the other side, at spindle assembly checkpoint, a cell can be arrested in metaphase if DNA damage is detected, DNA is not replicated completely, or chromosomes are not aligned on the metaphase plate, then it is unable to undergo the transition of the Finish checkpoint, thus sister chromatids remain unseparated and consequently the cytokinesis is not fulfilled. 6C). Most of the time during the cell cycle, the cells are in G1-phase with very slow max, additionally, low biomass yield on glucose (Table1) is observed as well. A description of the plasmid is given in Exercise 8. The diameter of averaged single cells exponentially decays from 10.2 m at 5C down to asymptotic value at around 8 m (Fig. 1. In the case of yeasts Saccharomyces cerevisiae, cells are dividing by means of budding and the formed cells are asymmetric in size: larger mother cell and smaller daughter cell (Figs 1 and 4). U6 RNA serves as a loading control. It is beyond the scope of this chapter to discuss in detail human mitochondrial diseases. From the chemostat experiment, it is well documented, that the macromolecular composition of the microbial biomass linearly depends on the specific growth rate (i.e. Saccharomyces cerevisiae (also known as Bakers Yeast or Brewers Yeast) is a unicellular fungus responsible for alcohol production and bread formation. Thus, the granularity, expressed as the SSC signal, is an integral parameter that exclusively includes both qualitative and quantitative aspects of the intracellular content; thus, the higher cytoplasm granularity, the stronger is side scatter of the laser beam. checkpoints) that ensure proper division of the cell (Porro etal.2009). It is usually found in the diploid form. 3, equation (4)). Saccharomyces cerevisiae is also present in labeneh, a strained yoghurt, as the predominant spoilage organism, reaching populations of 107cfug1 during refrigerated storage. Moreover, HSP104 RNA 3 ends are present at particularly low levels compared to HSP104 RNA 5 ends, suggesting the occurrence of a 35 degradation event. Length of the budding period (equation (7)). The ability to culture this yeast species as a haploid simplifies the isolation of mutants and haploiddiploid hybrids. \end{equation}, \begin{equation} In exponential phase, haploid cells reproduce more than diploid cells. Fundamental research on yeast mitochondria has assisted our knowledge of human mitochondrial function and disease. Saccharomyces cerevisiae occurs widely in foods but is infrequently designated as a causative agent for spoilage. WebCharacteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology and coverslipped, and filamentous structures were visualized with an optical microscope at 100X magnification. \end{equation}. The calculation has taken in account the fractional composition (single f1 and budding f2 cells) of the population at given growth conditions (equations (5) and (6), Table1; Fig. Change of dry weight of biomass was followed over time until it reaches saturation (|$C_x^{final}$|; exemplified at Fig. carbohydrate deposits in form of granules per cell volume, shape and size of the nucleus, amount and type of cytoplasmic organelles like mitochondria, vacuole, peroxisomes, cytoplasmic and membrane-associated ribosomes per cell volume, etc), i.e. Review Lab procedures for operating a Brightfield Light microscope B. 6E). As is true of CKII from other organisms, the native holoenzyme is tetrameric. Significance of S. cerevisiae in foods and beverages, Production of fermented beverages and breads, Production of food ingredients as a probiotic. Depiction of the holoenzyme as a heterotetramer of all four subunits, the linear form of the holoenzyme, and the specific order of subunits within the tetramer are consistent with available data (see text). {C_x} = N \cdot {V_{TV}} \cdot {\rho _x} WebSaccharomyces cerevisiae has been developed as a model eukaryotic organism for a number of reasons, for example: Saccharomyces cerevisiae is a small single cell with Meet bakers yeast - University at Buffalo 6F). Also, further to the discussion of storage and preservation of stock yeast cultures, RD mutants are difficult to store, and liquid nitrogen and 70C refrigeration have been found to be the most effective storage matrices (Russell and Stewart, 1981). (2009): the poor media yields a high level of asymmetry with large parent cells and very small daughter cells, whereas, in the rich media, parent and daughter cells are very close in sizes. DNA was stained with DAPI, and signals were overlaid with the Cy3 signal (bottom). The primary laser (argon-ion laser 488 nm) was used to record the light scatter by the non-stained cells. Thereby, if the max reduction is accompanied by increasing fraction of the non-budding cells in the population then it may indicate the fact that cells cannot pass through the G1-checkpoint until they fulfill mentioned above criteria. Temperature-induced change in cellular morphology (e.g. 1) (min/max). In general, the faster cells grow, the less granulated they are. 1A), which results in a fortiori wider width of the size distribution-peak (for more examples see Figs 3 and S1). 2, and the whole dataset was published in Zakhartsev etal.2015). so-called intracellular granularity. As with E. coli, various factors influence transformation efficiency (measured as transformants per microgram of DNA; also see Exercise 12, Introduction). Table I. G Selectivity of Mammalian G-Protein Activators, Christopher Buser, in Methods in Cell Biology, 2010. 6E). Assimilation of ethylamine, cadaverine, and lysine can differentiate these two species. This type of mutation is called petite because colonies (not individual cells) of such a mutant are usually much smaller than wild-type respiratory sufficient (RS) colonies (also called grande; see Figure 1). Biomass yield on glucose in substrate unlimited anaerobic batch, was reported in Zakhartsev etal. Therefore, it is expected that x can vary in dependence on max. 8). These variations in trehalose content, and the large amounts that can be accumulated, suggest that it plays an important role during the yeast life cycle. There is little quantitative data available on the occurrence of yeasts in minimally processed fruits; however, S. cerevisiae has been implicated as part of the spoilage flora of peeled oranges and commercially processed grapefruit sections. Diagram of the yeast mating pathway. 400x Studies correlating trehalose levels with the physiological and developmental activities of the cells have suggested that this disaccharide functions as an important carbon and energy reserve in starving cells (44, 45), in cells undergoing respiratory adaptation (46), in germinating spores (47), in vegetative cells during emergence from stationary phase (48), and in cells traversing the mitotic cell cycle under conditions of carbon and energy limitation (43). 3): (i) G1 or so-called Start and (ii) spindle assembly or so-called Finish (Chen etal.2004). Techniques used to measure HSP104 gene transcription can be found elsewhere and are not discussed further in this chapter (Birse et al., 1998; Jensen et al., 2004; Rougemaille et al., 2007). 4B). Saccharomyces cerevisiae CKII. The pRY121 plasmid used in this course serves as a useful instructional tool. it passes G1-checkpoint in the cell cycle and starts budding (Porro etal.2009). The cell flocculation/aggregation was not observed by optical microscopy at temperatures below 31C. Within 18.540C temperature range, the values of VTV and STS/VTV do not deviate from their corresponding asymptotic values, while exponentially deviate at temperatures below 18.5C. 1A), consequently, geometrically they are prolate spheroids. View publication. Dashed and shaded areas are 95% Confidence Intervals for corresponding regression curves. Correspondingly, the size of the bud was calculated as bud = 2 1.
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