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Sensors 2018, 18(12), 4089; https://doi.org/10.3390/s18124089
Monitoring Candida parapsilosis and Staphylococcus epidermidis Biofilms by a Combination of Scanning Electron Microscopy and Raman Spectroscopy
Institute of Scientific Instruments of the Czech Academy of Sciences, CZ-61264 Brno, Czech Republic
Biology Centre of the Czech Academy of Sciences, CZ-37005 Ceske Budejovice, Czech Republic
Department of Microbiology, Faculty of Medicine, Masaryk University and St. Anne’s Faculty Hospital, CZ-65691 Brno, Czech Republic
Author to whom correspondence should be addressed.
Received: 22 September 2018 / Accepted: 20 November 2018 / Published: 22 November 2018
The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.
Keywords:Raman spectroscopy; biofilm; sample preparation; scanning electron microscopy; cryo-SEM
Yeast and bacteria are microorganisms that can live as planktonic cells or in an organized formation called biofilm . During their adherence to surfaces or interfaces and their subsequent proliferation, the cells embed themselves into an amorphous extracellular matrix (ECM) , which is composed of extracellular polymeric substances (EPS) produced by the cells [3,4]. The presence of ECM is often considered an important characteristic of a mature biofilm . Among the main components of EPS is a mixture of polysaccharides, proteins, and extracellular DNA [6,7]. Since these substances are all highly hydrophilic, the biofilm water content can sometimes be as high as 90% of the total biofilm mass [1,8].
Microbes in natural ecosystems appear to have a pronounced tendency to colonize various surfaces and each other. The interest in such microbial communities—biofilms—has increased over the last three decades, because the biofilms are important in many aspects of health, biotechnology, etc. . The presence of biofilms can lead to human health problems, e.g., biofilm on teeth , also known as plaque and a factor in tooth decay and parodontosis, as well as the development of biofilm on medical devices such as catheters or implants . On the other hand, biofilms can be useful, as they are already extensively used in wastewater treatment [12,13] and play a role in biofuel production such as methane generation by methanogenesis  or in food production . Life in biofilms is favorable for microbes, bringing advantages such as enhanced persistence and resistance to environmental threats such as antimicrobial agents , toxic substances, thermal and oxidative stress [2,17]. Although the composition of biofilms varies depending on the system under study, in general the major component of a biofilm is water. Apart from water and the bacterial cells, the biofilm matrix is a complex formed principally by exopolysaccharides . Moreover, other macromolecules such as proteins, DNA and various products from the lysis of bacteria are present in the biofilm matrix . Studies of biofilms suggest that the biofilm matrix architecture is variable and it contains channels that enable water, nutrients and oxygen flow through the biofilm . However, the detailed architecture of the channels inside the ECM, and the processes operating within them have not yet been fully elucidated [16,20]. Therefore, in this study we have combined suitable microscopic and spectroscopic techniques that could be useful for studying the biofilms.
We examined the biofilm with emphasis on the differences in the apparent structure of the ECM, linked to various sample preparation protocols for SEM. The yeast Candida parapsilosis and bacterium Staphylococcus epidermidis have been studied in this project. These species are frequently found among the normal human microbiota [21,22]. However, in a medical context, the ability to form biofilms allows these microbes to colonize the surfaces of implants, consequently causing difficult-to-treat infections, especially in immunocompromised patients [23,24]. The presence of EPS protects the microbial cells from the natural defenses of the human immune system as well as from the effects of antibiotic treatments [25,26] and thus it complicates the therapy [1,8]. Understanding the biofilm structure can contribute to the research of biofilm formation and the underlying biochemical mechanisms. This will help to develop a more efficient treatment strategy for biofilm infections [27,28].
Microbial biofilms are usually investigated by various microscopic techniques including confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) [13,29,30], transmission electron microscopy (TEM) , Focused Ion Beam (FIB)-SEM  and by special SEM techniques, such as cryo-SEM or environmental-SEM [3,32,33,34]. The main limitation of the light microscopy techniques is the restricted magnification . This can be resolved by the use of SEM, which provides high-magnification images of the individual bacteria and yeast cells and their location and interaction within the ECM, which is important for understanding the morphology and physiology of biofilms [2,36]. However, a conventional SEM, where the sample is observed in high vacuum at room temperature, is limited due to the need for a dry sample . Biofilms are rich in water and the conventional sample preparation for SEM that includes desiccation as a prerequisite for imaging can cause substantial changes in the ECM and the microbial cell ultrastructure, leading to artifacts [5,34]. Chemical fixation with aldehydes and osmium tetroxide treatment help to preserve cell morphology and enhance contrast [38,39], while dehydration with ethanol or acetone series is used for the gradual replacement of the water inside the sample. However, this mode of preparation also causes some artifacts, such as cell membrane discontinuities , and it has other deleterious effects on morphology. In the case of cryo-fixation, the biofilm is not dehydrated but kept frozen to obtain high-resolution images closer to the native state of the sample [37,39,41]. It has been proven that in cryo-fixed biofilms, the bacterial ultrastructure preservation and the biofilm organization improved significantly . To reduce the damage inherent to these treatments, various innovative cryogenic sample preparation methods have been developed [41,43,44]. One of the simplest cryo-fixation techniques is plunging the sample into a liquid cryogen . In general, plunging into liquid nitrogen is not usually sufficient because of the Leidenfrost effect: a thermally insulating film of vaporized nitrogen forming around the sample, preventing fast cooling and allowing water ice crystals to form inside the specimen . However, cryogens like liquid ethane/propane are often used, for example in electron tomography, for fixation of very thin layers . Substantially more effective freezing can be achieved by increasing the pressure during exposure to the liquid cryogen. This can be performed by the high-pressure freezing (HPF) technique [47,48,49,50].
We coupled the SEM morphological examination of biofilms with chemical characterization by Raman microspectroscopy. Raman microspectroscopy employs a laser beam that is focused with the microscope objective lens in order to excite and collect Raman scattering from a small volume of the sample. Raman spectra from living microorganisms contain multiple spectral peaks corresponding to unique interatomic vibrations in biomolecules, e.g., nucleic acids, proteins, carbohydrates, and lipids [51,52,53,54]. It has been shown that Raman microspectroscopy and Raman imaging can be regarded as the methods of choice for many studies of microorganisms, cells and other biological samples [51,55,56,57,58,59,60,61,62,63,64,65]. Detailed databases of Raman spectral features encountered in biological samples had been published before . When characterizing biofilms using Raman microspectroscopy, the common approach is to analyze the biofilm as a whole. The spectra can be acquired point-by-point at selected positions or using line-scan techniques such as Renishaw StreamLine. In such cases, the Raman signal originates from the cells as well as from the ECM. Nevertheless, it may be useful to separate the ECM contribution from the Raman spectra, in order to fully understand the biochemical processes in the cells embedded in the biofilm matrix. It is well known that such cells express phenotypes that differ from those of their planktonic counterparts, i.a. the increased resistance to chemical treatments.
We employed SEM to study the ECM content and distribution in the biofilm, and the way it translates into its Raman spectral characteristics. The SEM images helped us to estimate the relative proportion of the ECM, which is in most cases ranges between 20% and 50% of the total biofilm volume. This means that in the Raman spectra, we observe the signal both from the bacterial cells and from the ECM, proportion of which depends on the growth stage of the biofilm. The proportion of the ECM increases with the age of the biofilm.
2. Materials and Methods
2.1. Biofilm Cultivation
Two biofilm-positive microbial strains that are often involved in serious infections [27,36,66] were selected as model organisms and examined in this study: the well-characterized ica operon-positive, biofilm and slime producing Staphyloccocus epidermidis strain CCM 7221 (Czech Collection of Microorganisms, Brno, Czech Republic)  and Candida parapsilosis BC11 from the Collection of the Microbiology Institute, Masaryk University and St. Anne’s University Hospital (Brno, Czech Republic) . The strains included in this study were stored at −70 °C in cryo-tubes (ITEST plus, Hradec Králové, Czech Republic). Prior to each experiment, the strains were thawed quickly at 37 °C and cultivated on Mueller-Hinton agar (Oxoid, Basingstoke, UK) at 37 °C for 24 h. The microbial cultures were re-suspended in a sterile physiological saline solution (PSS) to the optical density 0.5 of the McFarland scale .
In our experiments with the yeast biofilm, the wells of 24-well polystyrene tissue culture plates Nunclon (Nunc, Roskilde, Denmark) containing 1 mL of Yeast Nitrogen Base medium Difco (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) with 4% glucose (YNBg) and sterile substrate discs were inoculated with 100 μL of standardized cell suspension. Bacterial cultures were cultivated in 1 mL of brain-heart infusion (BHI) medium (Oxoid) with 4% glucose (BHIg) under the same conditions. We used standard cover slips or sapphire discs with the diameter 1.4 mm (No. 16706849, Leica Microsystems, Vienna, Austria) and 6 mm (No. 16770158, Leica Microsystems) as a substrate for HPF freezing and a cover slip (No. 1014/1818, Hecht-Assistant, Paris, France) for plunge freezing and conventional protocols for SEM; cover slips are widely used as a cultivation substrate for in vitro biofilm experiments [67,68,69]. After 24 h of incubation at 37 °C the substrate discs were removed from wells and further processed.
2.2. Conventional SEM
All the samples were imaged by several different high vacuum scanning electron microscopes (SEM) at room temperature. Specifications of the SEM devices and information on the imaging parameters are stated along with each procedure below in the text. Figure 1 summarizes all the sample preparation protocols for conventional SEM used in our experiments.
2.2.1. Method 1—Air-Drying (M1)
The simplest way to visualize the biofilm in vacuum of electron microscope is without any fixation of the structure. The microbial cultures of S. epidermidis and C. parapsilosis were cultivated in a medium for 24 h on a cover glass. Subsequently, the samples were air dried for 30 min. Imaging of the SE signal was performed by a VEGA TS 5130MM scanning electron microscope (SEM) (Tescan Orsay Holding, Brno, Czech Republic) at the acceleration voltage of 10 kV with the use of a homemade cathode lens with the deceleration voltage in the range around 3 kV and the working distance of 10 mm.
2.2.2. Method 2—Chemical Preparation (M2)
The microbial cultures of S. epidermidis and C. parapsilosis after 24-h cultivation under the same conditions as described in previous experiments were prepared according to the standard protocol [37,70,71]. Our samples were fixed in 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA), post-fixed in 1% osmium tetroxide (OsO4; Sigma-Aldrich) and thoroughly but carefully washed by PBS buffer (Sigma-Aldrich). The process of dehydration by ethanol (VWR Chemicals, Leuven, Belgium) series (30%, 50%, 70%, 80%, 90%, 95%, each step 15 min, and three times 100%) prepared the samples for drying. Here two methods of drying are compared. The first was done by hexamethyldisilazane (SPI-Chem, West Chester, PA, USA; HMDS; CAS 999-97-3) which was diluted with acetone (Sigma-Aldrich). Therefore, before applying this treatment it was necessary to replace the ethanol with acetone in four steps with increasing proportion of acetone (ratio ethanol/acetone 2:1; 1:1; 1:2; pure acetone) [37,70,71]. The second methodology for sample drying is the critical point drying (CPD) using CO2. The samples, prepared by conventional protocols [37,70] were coated by 10 nm of Au before imaging in a VEGA TS 5130MM SEM at the acceleration voltage 10 kV or in a Magellan 400L SEM (Thermo Fisher Scientific, Hillsboro, OR, USA) at 2 kV.
Our experiments with imaging at low temperature by cryo-SEM show the comparison of the biofilm structure at the same cultivation conditions but with different preparation protocols. We tested multiple cryo-fixation techniques; the workflow is summarized in Figure 1.
2.3.1. Method 3—Plunging into Nitrogen Slush (M3)
Freezing in the nitrogen slush was performed in the slushing station, a part of the ALTO 2500 cryo-preparation system (GATAN Inc., Pleasanton, CA, USA) [72,73]. The substrate for the cultivation of the biofilm samples was a cover glass of thickness 0.17 mm that was removed from the medium without any rinsing immediately before the freezing step. After freezing, the sample was transferred into an ALTO 2500 cryo-preparation chamber, perpendicularly freeze-fractured and subjected to a short sublimation at −95 °C for 3 min. The imaging was performed by SEM 7401F (JEOL, Akishima, Japan). The cryo-stage in the SEM was cooled at −135 °C, the anti-contamination aperture at −145 °C during specimen observation. The secondary electron (SE) micrographs were recorded at an electron energy range between 1 and 2 keV (low dose), and the working distance of approximately 6–8 mm over the fracture plane.
2.3.2. Method 4—Plunging into Liquid Ethane (M4)
Freezing in liquid ethane was performed in a homemade plunger. The substrate for cultivation of the biofilm samples was a cover glass of thickness 0.17 mm, which was removed from the medium without any rinsing immediately before the freezing step. After the freezing, the sample was mounted in a standard manner into a cryo-sample holder inside the nitrogen slush, then transferred into an ALTO 2500 cryo-preparation chamber, perpendicularly freeze-fractured and subjected to a short sublimation at −95 °C for 3 min. The imaging was performed by SEM 7401F (JEOL, Akishima, Japan) at the same conditions as described above.
2.3.3. Method 5—Cryo-Preparation by HPF (M5)
The high pressure freezing was performed by the HPF instrument EM PACT2 (Leica Microsystems) in standard conditions according to the instructions given in the operation manual. With the HPF EM PACT2, it is only possible to freeze sapphire disks with a diameter of 1.4 mm and therefore they were used as the substrate for the biofilm cultivation. Just as in the case of the plunging fixation the samples were not rinsed before freezing. After freezing, the sample was mounted into a homemade cryo-sample holder in liquid nitrogen , then transferred into a cryo-preparation chamber ALTO 2500, perpendicularly freeze-fractured and subjected to short sublimation at −95 °C for 3 min. Imaging was performed by SEM 7401F (JEOL) under the same conditions as described above. Our experiment with high-pressure freezing of the biofilm of Staphylococcus epidermidis was carried out by means of a HPF EM ICE (Leica Microsystems). In this case we used 6 mm sapphire discs as a cultivation substrate. After the freeze-fracturing in the ACE 600 cryo-preparation chamber (Leica Microsystems) the samples were sublimated for 5 min at −95 °C and then transferred by the VCT 100 shuttle (Leica Microsystems) into a Magellan 400L cryo-SEM (Thermo Fisher Scientific). The fractured structures were observed with a 2 keV electron beam at −120 °C and a working distance of around 7 mm.
2.4. Method 6—Combined Preparation: Chemical and Cryo-Methods (M6)
Our experiments with combined sample preparation started with biofilm cultivation on the cover glass under the same cultivation conditions as described above, followed by chemical fixation by 2.5% glutaraldehyde and 1% OsO4 in PBS buffer and thorough washing with PBS. In the next step, the samples were dehydrated by ethanol series (30%, 50%, 70%, 80%, 90%, 95% v/v ethanol; 15 min each, and three times with 100% ethanol) and frozen by plunging into the nitrogen slush. After mounting into a standard cryo-sample holder, the samples were transferred into a vacuum chamber (ACE 600) where they were sublimated overnight. The samples were then moved under high vacuum using a shuttle (VCT 100) into the SEM (Magellan 400L) and observed with a 2 keV electron beam at room temperature. Working distance was around 6 mm. The samples were metal coated by 2 nm of Pt.
2.5. Analysis of the Yeast Biofilm by Raman Spectroscopy
We used a commercial Renishaw Raman microspectrometer (Renishaw inVia, Renishaw plc., Wotton-under-Edge, UK), with a 785 nm diode laser as the excitation source. The laser beam was focused on the sample with a microscope lens (Leica, Wetzlar, Germany, 50×, NA 0.5), the laser spot diameter was 2 µm × 10 µm (note that this laser spot shape is characteristic for the Renishaw InVia instrument [51,64,65]).
The laser was focused on the surface of the biofilm, which was grown on CaF2 substrate. The samples were measured in two stages: first immediately after the cells were transferred to the substrate (no biofilm), and subsequently after 6 h of growth at 37 °C, when the fresh biofilm structures were formed. The spectra were measured for 30 s from different parts of the sample. The power of the excitation laser reached approximately 100 mW under the objective lens. The Raman spectra were treated with Savitzky-Golay filter to remove noise and with rolling circle filter for background fluorescence removal , and subsequently analyzed by principal component analysis . The software was written using MatLab (MathWorks, Natick, MA, USA).
3. Results and Discussion
3.1. Conventional SEM (M1 and M2)
SEM was found suitable for examination of the microbial biofilms, allowing visualization of microbial cell surfaces and the surrounding ECM with high resolution. Conventional sample preparation protocols for fully hydrated biological material involve following primary steps such as chemical fixation, dehydration and drying. This process modifies the biological material for the low pressure in the SEM chamber during the imaging. The advantages of a microbial biofilm specimen prepared in this manner are its stability, easy manipulation at room temperature, and the possibility of additional coating, which helps to visualize details in the surface structure. Contrarily, the use of chemical treatments and multiple rinsing steps exacerbates the artifacts, especially in the sensitive ECM structure and some parts of the biofilm layer may be completely destroyed.
The presented micrographs (Figure 2) show the comparison of influences of various preparation protocols on the microbial biofilm structure. The easiest way of biofilm preparation for SEM observation is air drying on a cover glass. In the micrographs (Figure 2a,b), the surface structures of the biofilm layer look similar for bacteria and yeast; the air drying caused a collapse of the three-dimensional structure. Moreover, in case of Candida parapsilosis we see that the extracellular space is filled by a dry matrix and the residues of the cultivation medium (Star in Figure 2b). It was not possible to visualize the clear surfaces of microbial cells (Square in Figure 2b) because they were covered by ECM and dry cultivation medium.
The chemical fixation and dehydration by ethanol series allowed preservation of the three-dimensional structure of the biofilm. However, in many areas of the cultivation substrate, the biofilm layer was destroyed and washed away due to the rinsing process during the sample preparation. ECM was partially preserved as a compact and roughened fiber-like matter in the extracellular space (Figure 2c–f, stars). The use of HMDS as a drying solution is a gentler way than CPD, the biofilm layer was less perturbed. Furthermore, the choice of the drying technique following the chemical preparation did not influence ECM quality. Therefore, drying by HMDS is a preferable method of the biofilm preparation for room temperature imaging by SEM whenever cryo-methods cannot be applied. The conventional SEM can be used for observing the surface of bacterial and yeast biofilm and obtaining high-resolution images of the spatial distribution of microbes. It can clearly be seen that the sample preparation represents a crucial parameter in the preservation of the three-dimensional structure of the biofilm. The influence of the chemical treatment is most obvious on the spatial architecture of the biofilm and the compact structure of the ECM. The significant disadvantages of the chemical preparation are the partial loss of the biofilm from the cultivation substrate and the time requirements for this process.
3.2. Cryo-SEM (M3, M4 and M5)
The use of low-temperature preparation by cryo-SEM for observing biological samples brings a number of advantages compared to conventional SEM at room temperature. The samples do not have to be fixed by chemicals treatment and rinsed several times by a buffer. Moreover, the dehydration series is eliminated and the lengthy drying is no longer needed, therefore the artifacts associated with these processes are eliminated. On the other hand, the observation of biological structures in SEM at low temperatures requires specialized equipment and has its limitations and drawbacks. It is well known that suboptimal freezing speed during the cryo-fixation causes disruptions to the soft hydrated material due to the water ice crystallization . Therefore, the choice of a particular cryo-fixation technique is crucial to obtain unperturbed structure of the frozen specimen.
The comparison of the freezing methods for yeast and bacterial biofilms is the main aim of this section of our study. The perpendicular freeze-fracture of the samples (24 h old biofilms of Staphylococcus epidermidis and Candida parapsilosis) frozen by plunging into nitrogen slush are shown in Figure 3a,b.
The inner structure of the microorganisms (Marks—squares in Figure 4) seems to be sufficiently preserved in the range of magnification around 5000×. The thickness of the biofilm layer is approximately 10 µm. The structure of the ECM containing the extracellular biopolymers and the cultivation medium has a sponge-like character. We consider this structure to represent a micro-segregation (±0.5 µm) caused by ice crystal growing during the slow freezing process. The method based on plunging the sample into liquid ethane should be appropriate for freezing a sample with thickness up to 10 µm . In our micrographs the final structures after fixation by plunging into liquid ethane look very similar to previous experiments, probably due to the existence of a cultivation substrate such as described in literature . Nevertheless, the use of sapphire discs as a cultivation substrate for plunge-freezing seems to be more suitable from the perspective of their thermal conductivity. However, several experiments show that the plunge freezing fixation of cells cultivated on sapphire discs lead to ice crystal segregation as well ; this result also corresponds to our experiments. Noticeably better preservation of the whole microbial biofilm, including the extracellular matrix, is clearly visible in Figure 3e,f (marks—stars). High-pressure freezing ranks among the cutting-edge sample preparation techniques for cryo-SEM. Besides the fast cooling rate, a positive influence is apparently brought by the use of sapphire discs with better thermal conductivity compared to other substrates. The structure of ECM (Figure 3e,f, marks—stars) is very smooth within the magnification used and we were able to detect only minimal disruptions due to the ice crystallization. Moreover, it is possible to recognize the denser parts of the biofilm that can be observed in the surroundings of the microbial cells .
Cryo-methods in SEM are capable of providing information about the ultrastructure of the microbial biofilm interior, assuming that freeze-fracturing is applied. It is possible to visualize the areas where microbes adhere to the surface of the cultivation substrate and the contact fields between individual microbial cells. On the other hand, examination of the biofilm surface (Figure 3, marks—circles) could not be performed, because the biofilm layer was covered by frozen liquid (the cultivation medium with a content of EPS).
3.3. Combined Preparation—Chemical and Cryo-Methods (M6)
Alternative sample preparation protocol is the combination of chemical fixation by solution of GA and OsO4 and dehydration by the ethanol series such as in the case of biological sample preparation for conventional SEM techniques by the standard chemical method. When the total water content was replaced by 100% ethanol, the samples in our experiments were frozen by plunging into the nitrogen slush, and transferred to the cryo-preparation chamber where they were sublimated overnight until they were completely dry. The sublimation process started at −140 °C, then temperature increased to −80 °C with a speed of heating 4 °C/min for 6 h and finally samples were left to warm up to the room temperature spontaneously. Additional coating (4 nm, carbon) allowed a production of high-magnification micrographs of our biofilm samples. From our results it can be concluded that the M6 protocol is the most favorable means of biofilm preparation with the aim of surface visualization. The micrographs in Figure 4 show the spatial distribution of microbes and ECM similar to conventional preparation for room-temperature SEM. The ECM (Figure 4, marks—stars) of the bacterial biofilm seems to have an aggregation character which can arise from the chemical treatment during the preparation. The fiber-like structure of the extracellular matrix that covered and interconnected the yeasts bodies looks very similar to the ECM in samples prepared by CPD, but biofilm samples were washed away to a lesser extent due to the absence of chemical drying or CPD. We were not able to detect any evidence of a gel-like matrix covering the microbial cells as described in many publications [1,24,78]. This is to be expected, because the whole freeze-drying procedure was applied in this method. We found that the visualized structures are the remnants of the cultivation medium and the condensed matrix components. The limitations of the six different preparation techniques for SEM on bacterial and yeast biofilms (M1–M6), the benefits and influence on the biofilm structure are summarized in Table 1.
3.4. Analysis of the Yeast and Bacterial Biofilms by Raman Spectroscopy
We assessed the chemical composition of the biofilms using Raman microspectroscopy. Our central objective was to identify the main components of the biofilm. We compared the Raman spectra from the freshly inoculated substrate, containing only the microbial cells, but no ECM, with relatively fresh (6 h old) biofilm, containing a definite proportion of the ECM, see Figure 5. To this end, we assigned the individual peaks of the measured spectra, see Table 2, and we employed PCA to pinpoint the differences in the chemical composition, see Figure 6. The associated PCA loadings reveal the essential changes associated with the ECM production, see Figure 7. We found the main difference between the freshly inoculated substrate and 6 h old biofilm is in the production of proteins, sugars, and lipids (peaks 6, 7, and 8 on Figure 5). This stems from the generation of various polysaccharides, proteoglycans, lipopolysaccharides and lipoproteins as the basis of the ECM.
While the differences in the spectra of Candida parapsilosis at 0 h and 6 h are very pronounced, especially in the region 1340–1360 cm−1 and 1456 cm−1, Staphylococcus epidermidis spectra at 0 h and 6 h show only marginal differences (Figure 5), although they are clearly visible with the aid of the PCA analysis (Figure 6 and Figure 7). This discrepancy is probably caused by the differences in the optimal conditions for biofilm production by the two studied species. Candida parapsilosis, which is eukaryotic, may have developed more complex strategies to react to suboptimal or stressful conditions in general, including the ability to generate biofilm more quickly and efficiently, compared to Staphylococcus epidermidis, in which the biofilm production may be linked only to a specific set of growth conditions. We conclude that while we were able to stimulate the biofilm growth in Candida parapsilosis, in the Raman experiments we did not meet the optimal conditions for biofilm production in Staphylococcus epidermidis. However, it is obvious from the SEM observations, that given enough time, the biofilm growth around the Staphylococcus epidermidis cells is abundant.
The chemical changes associated with the formation of biofilm can be easily observed in the PCA loadings in Figure 7. While the Candida parapsilosis biofilm appears to be composed mainly of fungal exopolysaccharides that lack nitrogen in their structure (peaks at 1340–1360 cm−1) [60,79], the biofilm of Staphylococcus epidermidis is relatively richer in nitrogen containing polysaccharides such as poly-N-acetylglucosamine (peaks around 1400 cm−1) [60,80]. The ECM of Candida parapsilosis is composed of several groups of chemical components, including presumably the lipoproteins and proteoglycans. In contrast, the ECM of Staphylococcus epidermidis appears to be more chemically uniform. This uniformity apparently stems from the relative simplicity of the bacterial (prokaryotic) metabolism compared to the eukaryotic metabolism of Candida parapsilosis.
In order to study the microbial biofilm structure of Candida parapsilosis and Staphylococcus epidermidis we have investigated different sample preparation techniques for SEM. The effect of sample preparation for conventional SEM allowing surface imaging at room temperature was compared with cryo-SEM techniques employing plunging into various liquid cryogens and HPF. For the cryo-SEM imaging of the biofilm inner structure we have selected the freeze-fracturing technique. We made a comparison of applied techniques for microbial biofilm studies which indeed showed different influences on the final structure of the biofilm. Based on our findings the best candidate for biofilm evaluation can be selected.
We showed that a combination of Raman spectroscopy with selected SEM techniques can provide a deeper insight into the chemistry and composition of biofilms. Such studies involving the influence of variations in the amount of extracellular material during the different stages of biofilm growth are currently under way in our laboratories, making use of a combination of SEM and Raman spectroscopy. We believe that the detailed view of the biofilm structure and composition can advance the better understanding of biofilm structures.
Conceptualization, K.H., V.K., J.N., F.R. and O.S.; Methodology, K.H., V.K., J.N., and O.S.; Validation, K.H., J.N. and O.S.; Investigation, K.H., V.K., and O.S.; Resources, F.R.; Data Curation, K.H., V.K. and O.S.; Writing-Original Draft Preparation, K.H., V.K., O.S., and Z.P.; Supervision, V.K. and O.S.; Project Administration, V.K.; Funding Acquisition, V.K.
The research was supported by the Czech Science Foundation (project 17-15451S), the Ministry of Health of the Czech Republic (projects 16-29916A and 16-31593A) and the Ministry of Education, Youth and Sports of the Czech Republic (project LO1212). The research infrastructure was funded by Ministry of Education, Youth and Sports of the Czech Republic and European Commission (projects CZ.1.05/2.1.00/01.0017 and LM2015062 Czech-BioImaging) and by the Czech Academy of Sciences (project RVO:68081731).
K.H. is grateful to a FEI/CSMS scholarship for support. K.H. and V.K. thank Roger Wepf for helpful discussions regarding cryo-SEM.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1. Diagram of microbial biofilm sample preparation for SEM.
Figure 2. SEM micrographs show the comparison of preparation protocols for microbial biofilm structure (in the left column S. epidermidis and in the right column C. parapsilosis): (a,b) no chemical preparation and air drying; measurement parameters: 10 kV with the use of a homemade cathode lens with the deceleration voltage in the range around 3 kV, WD 10 mm; (c,d) chemical sample preparation and drying by CPD; measurement parameters: (a) 10 kV or (b) 2 kV, WD 8 mm; (e,f) chemical sample preparation and drying by HMDS. Marks: stars—ECM, squares—cells; measurement parameters: 2 kV, WD 8 mm.
Figure 3. SEM micrographs showing the comparison of preparation protocols applied to microbial biofilms (in the left column S. epidermidis and in the right column C. parapsilosis): (a,b) plunge freezing into nitrogen slush; (c,d) plunge freezing into liquid ethane; (e,f) freezing by HPF. Marks: stars—fully hydrated ECM, squares—microbes, dots—surface of biofilm layer; measurement parameters 1 to 2 keV and 6–8 mm.
Figure 4. SEM micrographs show the comparison of the bacterial and yeast biofilm structure after the combined sample preparation: (a,b) S. epidermidis; (c,d) C. parapsilosis. In the left column, images are displayed in a lower magnification, while the details are shown in the right column; measurement parameters: 2 keV, WD 6 mm.
Figure 5. Raman spectra of biofilms. (A): Candida parapsilosis; (B): Staphylococcus epidermidis. Comparison of the freshly inoculated substrates containing no ECM (0 h, blue) with 6 h old biofilm, showing the start of the ECM production (6 h, red). The Raman peaks associated with biomolecules are numbered, see Table 2 for the assignments. The spectra were averaged from six separate measurements.
Figure 6. PCA plots for the two species (A) Candida parapsilosis and (B) Staphylococcus epidermidis. The clusters of spectra are associated with the two incubation times. Blue squares/0 h—initial cultures with no ECM; red diamonds/6 h—6 h old cultures with starting ECM formation.
Figure 7. PCA loadings for the two species (A) Candida parapsilosis and (B) Staphylococcus epidermidis. The main differences between the cells that did not yet formed the biofilm and the ones that already produce it are presented mainly in the quantity of the generated proteins, sugars and lipids.
Table 1. Comparison of benefits and limitations of sample preparation protocols for SEM (Methods M1–M6) and their influence on biofilm structure as shown in schematic drawings. Our best candidates for sample preparation techniques are labelled green.
|M1—air-drying||Speed of sample preparation |
Repeatability of measurement in SEM at room temperature
Suitable for surface imaging
|The loss of the 3D structure |
Deformation of microbial biofilm
Deformation of ECM The possibility of imaging only the sample surface (not interior)
|Deformation of biofilm |
|M2—conventional chemical preparation||Repeatability of measurement in SEM at room temperature |
The 3D structure is preserved.
Suitable for surface imaging
|Long-term procedure |
Damage of soft biofilm sample due to multi-steps washing
Artefacts with chemicals treatment (the change of gel-like ECM into fiber structures)
The sample surface imaging
|Biofilm is washed out |
|M3—plunging LN2; cryo-SEM||Speed of sample preparation |
The 3D structure of microbial cells is preserved
Possibility of biofilm interior imaging (used freeze-fracturing technique also suitable for M3–M5)
|Artefacts with freezing procedure |
Freezing is sufficient for very thin samples
Limitation for surface imaging because of water content in biofilm samples
|“Large” ice crystals |
|M4—plung. Ethane; cryo-SEM||Speed of sample preparation |
The 3D structure of microbial cells is preserved
Possibility of biofilm interior imaging
|Artifacts with freezing procedure (smaller ice crystals inside biofilm than by M3) |
Freezing of thin samples
Limitation in surface imaging because of water content in biofilm samples
|“Small” ice crystals |
|M5—HPF freezing and cryo-SEM||Speed of sample preparation |
3D structure of microbial cells/ECM is nicely preserved
The best freezing technique for samples with thickness up to 200 µm (exp. tested)
Biofilm interior imaging
|Limitation in surface imaging (water content in biofilm) |
Limitations connected with HPF machine–cultivation substrate (sapphire discs for freeze fracturing; Al or Cu-gold discs)
|Optimal prep. of biofilm for interior imaging |
|M6—Combined preparation||Speed of sample preparation |
3D structure of biofilm
Biofilm surface imaging
Repeatability of measurement in SEM at room temperature after freeze-drying
Less washed out biofilm
|Artifacts from chemical fixation (the change of gel-like ECM) |
The imaging of sample surface
|Applicable prep. of chemically fixed biofilm for surface imaging |
Table 2. Assignments of Raman peaks of C. parapsilosis and S.epidermidis biofilms .
|No.||Wavenumber [cm−1]||Peaks Assignment|
|1||1002||Symmetric-ring breathing of Phe|
|2||1033||C-H in-plane stretch of Phe|
|3||1065||C-C stretch of lipids|
|4||1125||C-N stretch of proteins|
|6||1267||Lipids, Amide III|
|8||1456||CH2 scissoring, Lipids|
|9||1660||Amide I, Lipids|
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

domain: chemistry


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After speaking with many people and reading a lot of forums, I’ve found that, while general anxiety may be something most of us can “cope” with, whether it be through a prescription, holistic medicine, or mental and physical practices, there are times when panic attacks momentarily throw all that coping out the window. Email firstname.lastname@example.org , or give us a call at (860) 348-3376, and you can record your story in your own words. Your message of support will help lift the mood of both that person, the victim of illness, and their family. Now that you know that anxiety isn’t just overblown nervousness, you’re not going to tell your anxious loved one to “Get over it”. Most Downloaded Pictures of The Month. You’re not going to tell them to calm down or call them weak. So much so, sometimes they might be the ones to ruin a relationship. We are blessed that God has already given us many comforting words through the Holy Spirit-inspired words of the Bible. “You can’t always control what goes on outside. If you have someone on your holiday shopping list who you suspect is struggling with anxiety this year, there are a few things you should think about before selecting their gift. Even better, do a few group activities. Be compassionate with the person, before, during, and after their attack. They often have no idea what anxiety is like, or how to care for someone who is deep in its throes. The content of this field is kept private and will not be shown publicly. If your partner has PTSD, here are some things to remember. They need your understanding, forgiveness, and patience more than you know. Then just keep doing that every day. If you don’t know what to say, here are 25 different examples of encouraging words for family of sick person. Being scared means you’re about to do something really, really brave.” – Mandy Hale “Anxiety was born in the very same moment as mankind. "Fear itself is something we all experience, so when talking to someone with anxiety it's really all about just connecting with them." Anxious people can be irritable. Talking to your loved one about the accident or comforting them can be difficult. These emotions can overwhelm us and keep us paralyzed. It also helps the feelings feel valid, which helps eliminate increased anxiety because a person might feel like they are being stupid or the feelings are wrong. DON'T let anxiety … Ask questions. For "Holistic Healing for Anxiety" a 28-day online course, click here: Can This 6-Second Trick Prevent a Panic Attack? Positive Quotes For A Sad … You want to be a beacon of hope, not of tension. 2. How Many Years of Life Will a Bad Relationship Cost You? I have aniexty & depression and that is very calming. If encouraged, it cuts a channel into which all other thoughts are drained. In allowing someone to express their feelings, it might even allow you, the comforter, to gain insight into their world and help you gain further understanding of what they are going through -- even if you don't have an immediate solution. 1. Example Words of Encouragement Card Messages. “P.S. 4. There are people in this house who need to sleep!” Dad is a comfort only if the child knows him to be consistently caring and willing to use his greater strength for the benefit of the child. But it can be hard to know the right thing to say to a friend or family member in such a vulnerable moment. As someone who has depression these are the things u should say : 1. its not my fault yes I know I am depressed the last thing I wanna do is be reminded it's my fault just don't say this 2. Use words of affirmation and do not try to downplay a person’s emotions when they get anxious even when their reaction(s) does not make sense to you. For me, hearing, ‘I’m excited to see you tonight!’ or ‘I love you and I’m proud of you,’ every so often can go a really long way in making me … Depression, anxiety, loss of interest, or irritability are all moods that can indicate someone is considering suicide. Instead say: “I’m always here for you.” Comforting Someone with an Anxiety Disorder. “The largest part of what we call ‘personality’ is determined by how we’ve opted to defend ourselves against anxiety and sadness.” —Alain de Botton It is a business asset. Getting to know how you can help, listening to exactly what the anxiety sufferer says, and being willing to implement that help can be a wonderful gift to someone who may feel invisible because of their illness. Go for a drive or play a board game. Bible Verses About Worry and Anxiety - Find comfort and peace through Scripture that promises you hope and a future. Still other forms of anxiety will require medication. Here are some examples of comforting words to think about the next time you’re struggling with how to help someone going through tough times. It is the best antidote for anxiety and depression. That means almost one in five people around you experiences anxiety for longer periods of time, at higher levels than most people and in … Here are some helpful things to say to someone who is struggling. It can emotionally and physically cut you off from your support structure through an intense aversion to situations that are, for most people, perfectly normal. Many viewers commented on what their personal experiences have been and how someone’s words ... (2015). There ARE ways to help ! “Do not let your difficulties fill you with anxiety; after all it is only in the darkest nights that stars shine more brightly.” – Ali Ibn Abi Talib “It’s OKAY to be scared. A variety of anxiety quotes to help sooth your anxiety, or just help you understand that you aren't alone in your anxiety. “I’m Always Here if You Need to Talk” Sometimes a person with anxiety will want to … In hopes that you may find these helpful, below I’m sharing some Bible passages that I’ve found very comforting when I’ve struggled with anxiety … I do this for a couple of reasons. Be the reason someone feels seen, heard, and supported by the whole universe. According to experts, the one word you should never say to someone with anxiety is "relax." Let’s find out as we take a look at 7 ways you can comfort someone with anxiety. To help someone with anxiety, you must first learn what it actually is. Top ten things to say to someone with anxiety. They may be struggling with extreme depression, feelings of isolation, the effects of complex trauma or abuse, mental health issues, worthlessness, shame and an overwhelming desire to end the pain they’re feeling.” 18. Those who suffer from anxiety may feel alone in their disorder. Anxiety & Panic. 6. Anxiety 22 Calming Quotes for People with Anxiety Sometimes it takes only a sentence or two to get on the other side of anxiety. Comforting Words For Someone With Depression. Depressed Quote. Just enter your email below. 5. Telling someone with anxiety that ‘everything will be fine,’ can fall on deaf ears since their tendency to believe it is nil. They can seem irrational, misjudging your remarks and expressions as being more negative than you meant. Get these activities in before the sun sets on summer! 3. Be ready with the swatter. Religious OCD: When Faith Becomes Obsession, Click here for our full list of newsletters. This decidedly uncomfortable conversation may just end up being the biggest comfort of all. Positive reinforcement will associate good feelings with healthy actions. Anxiety can make people feel lost and alone. It attracts and keeps friends. For people who have illness anxiety, trying to self-diagnose by researching their symptoms turns into a source of many new questions, concerns, and fears. Each person’s illness is unique, and the circumstances surrounding their situation are just as varied as their depression is. “Good humor is a tonic for mind and body. But never punish someone for their anxiety—it will only make things worse. The symptoms of anxiety cause physical and emotional problems that lead to more anxiety, which creates a snowball effect that can spiral out of control unless help is sought. 5. If you lose the gig, the lover, the house, you’ll still be okay. Posted Sep 17, 2014 Receive a Daily Measure of God's Word and guidance straight to your inbox. 8. I am a master at covering up my anxiety when I’m around other people. Who Most Wants to Get Back Together With an Ex? What’s the simplest thing you can do to help comfort someone with anxiety? 9. You are strong and capable When you find out someone is depressed, it’s tempting to attempt to immediately try and “fix” the problem. Final Thoughts On Some Things To Avoid Saying To Someone Suffering From Anxiety If you’re trying to help someone with anxiety, you shouldn’t be saying the aforementioned 20 things. Think positive thoughts, make the most of your time with us, and keep looking forward towards better days. This page was created by our editorial team. It is a disease caused by changes in the brain. Here's how to help someone having an anxiety attack. Read on for more expert-backed tips on what not to say to someone who's grieving. They didn’t think it possible, but HERE YOU ARE, providing them hope and helping them envision a future they knew they wanted but didn’t believe was possible. “Smile, breathe, and go slowly.” —Thích Nhất Hạnh It lightens human burdens. Jul 15, 2012 - Explore Frances Pierce's board "Comforting Words" on Pinterest. Storms don’t last forever. “Climate anxiety is combined in people’s lives with other anxieties,” writes Dr Pihkala, “such as those related to choosing a profession.” In case something else is causing deeper anxiety in a person’s life, he argues, climate anxiety should be taken seriously to better evaluate the overall situation.

domain: psychology


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SYDNEY, AUSTRALIA, August 8, 2014 (Sydney Murugan Temple): The Saiva Manram & World Saiva Council Australia Inc. jointly present the Sydney Murugan Saivaneri Conference 2014. The theme of the conference is Humanitarianism in Saiva Agamas, Thirumurai and Siddhantha Texts. the conference will be held on August 29, 30 & 31, 2014, and tutorials on August 23, 24 and September 6 & 7, 2014. All conference sessions, religious ceremonies, cultural event, and tutorials will be held at the Sydney Murugan Temple Complex, 217 Great Western Hwy, Mays Hill NSW 2145, Australia. The conference is billed as a wonderful opportunity to learn the intricacies and greatness of Saivam.
For more information contact firstname.lastname@example.org.

domain: news


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Golden Oak Wood Flooring as Your Reference » Truss floors are simply just that. These are made of small dimensioned lumber, interconnected in a webwork pattern through metal or wood plates. Occasionally, the trusses will be built on site, utilizing plywood plates to hook up the webwork alongside one another. Generally they are simply installed 24″ aside, either suspended on bearing surfaces or beams, or installed with plywood trim or rim joists surrounding the perimeter. Strapping is installed on the bottom side, Golden Oak Wood Flooring to avoid submiting place, which really is a common ailment for profound truss components. In the case of long span truss work, bearing measures of at least 3″ are quite common. Trusses span greater distances than framed floor assemblies and can be made to span the entire building, eliminating center load bearing aids. They are reasonably more costly than framed floor assemblies, but provide a remarkably strong floor with little deflection or “bounce” to it.
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Golden Oak Wood Flooring as Your Reference » The made joist, which really is a relatively new product, is often made from low priced materials in the shape of I beam, comparable to material beams in bigger buildings. This implies that the joist is designed with a thicker top and bottom level border, and generally interlocking aspenite vertically spanning between your two. These systems are incredibly strong, often with the capacity of spanning the complete width of the building. One Golden Oak Wood Flooring downside is that kind of floor requires special hanger systems designed for the joists, to enable these to be hung from one another or against beams/bearing surfaces. Manufactured joists have become a popular flooring system, for they are relatively cheap, reduce labour time and offer satisfactory support. However, contractors need to familiarize themselves with its installations, for poor assembly can cause severe structural damage to the joists. A good example is a three point, centre bearing joist, remaining with the very best chord uncut, which can potentially fail or yank apart, over the guts bearing point.

domain: design


Here’s some footage of Michigan St. shooting drills
I can’t say this was a greatly played game because it most certainly wasn’t that but it was fun to watch. A couple of teams that let it all out there, diving all over the place. Easily the grittiest game I’ve seen in a while. Syracuse the big underdog did what they always do and that’s a play a 2-3 zone that apparently nobody can figure out. Yeah, their players are long and all that but when you go and shoot 17-66 from the field, you’re gonna have a bad time. I repeat, Michigan St. went 17-66 from the field. They only made 17 shots and missed 49 shots. That’s amazing especially for a team that several analysts said was a final four team. I get that in the tournament that anyone could lose but missing 49 shots? That’s wild wild stuff. What was also nuts was that it seemed that Michigan St. had 48 points for about a year. That might be a slight exaggeration but only by a little bit. It just seemed that Michigan St. were just passing around the perimeter over and over again, never quickly reversing the ball and when they did get the ball inside to the highpost their big man acted like he never held a basketball before and frantically threw it back out to the wings. Syracuse moves on to play Duke and I have no idea how that game goes. Easy to say that the Orangemen get waxed by Duke who will be able to push the ball much better than the Spartans but again, that zone is like a Indus Valley Citadel. No one gets past it. As for Michigan St., I’m sure they will be a top team next year but they might want to hit the gym and watch some form shooting videos or something. I don’t know, today was ridiculous with all the missing.


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domain: artistic


- 5 Dec 2019 8:04 AM
- Budapest Business Journal
The 70-year-old company has been involved in virtually all the major railway electrification projects in Hungary over the past seven decades, and so has deployed more than 3,000 km of overhead line for the railway network in Hungary.
The company’s revenue exceeded HUF 16 billion last year, and as a result of technological advances and asset purchases in recent years, its technical capacity allows it to reconstruct or build up to 200 km of overhead line from its own resources.
“The proportion of electrified railway lines is still significantly below the European Union average of 53.7%, but the investments made in recent years bring us step by step to complete the electrification of all high-traffic or internationally connected Hungarian railways,” said Zsolt Homlok, majority owner of Vasútvill Kft.


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domain: entertainment


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Have you noticed that milky haze across the sky? That’s smoke in the upper atmosphere from Western wildfires. There are 80 fires burning across 13 states and the smoke from those fires reaches across the entire US. Check out this NY Times story, See How Wildfire Smoke Spread Across America. (MVCC students and staff can sign up for a free account on the NY Times through our library).
Since President Biden just signed the law making Juneteenth a federal holiday, here is a video from our library from 2019 about the Juneteenth holiday.
Talk Description: “Moraine Valley Community College students will learn about the oldest known holiday to commemorate the ending of slavery in the United States. The session will focus on the historical significance and cultural traditions of the forgotten holiday.”
Check our this amazing visual guide to understanding comics! Candida Rifkind, Brandon Christopher, and Alice RL’s “How Comics Work”.
Our library provides access to 1000s of graphic novels and comics (in both print and digital options). Sometimes getting started in this medium is the tough part, so that’s why “How Comics Work” is a great guide. In just a few pages, it gives you the short and to understand this iconic popculture literary form.
Dr. Alexander Alonso presents a talk on Nephrology which focuses on the functions of the kidneys. He talks about his path to this unique profession and the benefits of the job.
In the new iPhone update (14.5), iPhone users will have the ability to “Ask App not to Track.” This is a new feature with this update that makes your online privacy stronger. For more information, take a look at this Washington Post article: Facebook now has to ask permission to track your iPhone. Here’s how to stop it. (Note: enter “Moraine Valley” as your institution and then use your campus login to read it.)
The new iPhone update 14.5 will be pushed to phones over the next several weeks.
We celebrate National Poetry Month by featuring student poets. We also have student musical guests. As part of this event, we announce the winners of the student poetry contest that was organized by the Glacier and the Library.
Join psychology professors Mitchell Baker, Dr. Laura Lauzen-Collins, and Nick Shizas as they explore psychology concepts that help us better understand the rise of extremism and belief in conspiracy theories.
Join history professors Merri Fefles-Dunkle, Josh Fulton, and Jim McIntrye as they explore the role of the state throughout history to help us understand the connection between pandemics and the rise in extremism, mistrust, and rejection of authorities. This event is organized by the MVCC Democracy Commitment.
Faculty members discuss the biology, public health aspects, and the psychological factors around the Covid-19 vaccines in the United States. This panel is organized by the MVCC Library. Panel members include: Judy Corcoran, Nursing Laura Lauzen-Collins, Psychology Peter Porter, Biology The discussion is moderated by librarians Hannah Carlton and Troy Swanson.
Don’t miss our 2020 panel “Racial Health Disparities: Equity in the Light of Covid-19“

domain: library


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Li ne crusher in cement plant. Home li ne crusher in cement plant Coal Powder Processing Ground Calcium Carbonate(GCC) Ground Calcium Carbonate(GCC) semi-mobile or mobile crushing plants with plant capacities of . . Cement Production Line.500th Large Limestone Production Line. Limestone is mainly composed of calcium carbonate (CaCO3), MO's Hardness 3 degrees. The limestone particles or powders can be used in building materials, road construction, metallurgy, chemical and other industries after crushing or grinding.Dec 04, 2021 Calcination The limestone (CaCO 3) is heated in a rotary or shaft kiln where it breaks down into calcium oxide, i.e. quicklime (CaO), and carbon dioxide (CO 2). Crushing The quicklime is reduced into different sizes. Grinding The crushed quicklime is ground into a.
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Ground Calcium Carbonate Processing. . Ground Calcium Carbonate(GCC) A hammermill is a mill whose purpose is to shred or crush aggregate material into smaller , Invention, involved conversion of rotary water wheel energy to linear trip hammer energy, around Zhao dynasty period of 4th century in China. 110V Electric Hammer Mill.Calcium carbonate powder production line 1. sand and stone supplier pretoria. stator rotor pigment grinding machine. hoisting technology in mining. Cone Crusher for sale tulsa Ok. soapstone grinding machines. copper ore crushing system in the republic of guatemala. iron ore crusher mining crusher in the sea of indonesia.Calcium Carbonate Limestone Machine. Calcium carbonate limestone machine production line is the preparation of a pipeline of heavy calcium, heavy calcium powder particle size of the finished production line preparation, wide sales market, much of the downstream users, to investors bring considerable benefits, the production line equipment a reasonable selection or.
It has comprehensive mechanical crushing propertiessuch as rolling.grinding and . Contact. various Industries Demand for Calcium Carbonate Powder . 2021-3-1 various Industries Demand for Calcium Carbonate Powder and 1500 Mesh Grinding Mill Price. At present, some calcium carbonate powder making enterprises are still stuck in the traditional and.Feb 16, 2021 Calcium Carbonate Limestone Machine - Crusher Application. Calcium carbonate as a main component of white powder. Calcium carbonate is a very wide range of applications, the most common is for the construction industry, which is the industrial use of calcium carbonate the other is a food grade calcium carbonate, as a common calcium.The whole Calcium carbonate Powder Production line can be divided into four parts as follow. Crushing Usually, the Calcium carbonate to process usually have such big sizes that it is impossible to be used to grinder powder due to the limited size of the input opening.So big stones have to be crushed into the centimeters.
Daswell Ground Calcium Carbonate Process - CaCO₃ Ground calcium carbonate, commonly referred to as GCC, is widely used as an industrial mineral.Ground calcium carbonate process refers to the ground calcium carbonate manufacturing or production process, in which raw materials are ground to fine and even ultra-fine ground calcium carbonate powder.Calcium Carbonate Grinding Process the choice of the milling equipment directly determines the status of the production of calcium carbonate in the entire production line but also determines the quality of the finished product of calcium carbonate powder 171 Limestone Crushing Production Line Iron ore processing and mineral.If the powder calcium carbonate fails to achieves the required fineness by analyzer, it will return to the grinding chamber to continue to grind then through analyzer, calcium carbonate is put into whirlwind powder collector finishing separation and collection. Lastly, with industrial filter cloth isolated exhaust was put into powder at one time.
Calcium Carbonate Ball Mill Micron - MTM Crusher. Calcium Carbonate Crusher Plant In India. Currently a lot of calcium carbonate product name, called mixed, there is no into calcium carbonate particles, and then by . limestone crushing into calcium carbonate powder less . limestone crushing into calcium carbonate powder less than.2021-2-16 Machinery Used In Calcium Carbonate. Machine Used In Precipitated Calcium Carbonate Plant. used calcium carbonate plant used calcium carbonate plant XSM excellent mining crushing machinery products or production line design the company is committed to building the Chinese brand mine crushing and processing machinery mainly crusher.The working flow of vortex mill coating machine for calcium carbonate powder is simple. First of all, calcium carbonate and modifier need to be sent to a screw conveyor for mixing after weighing. Then, the mixed materials are sent from the hot air stove to the vortex mill powder coating machine for high-speed stirring and coating.
Li ne quarry selection sand washing machines supplier Calcium Carbonate Powder Production Stone Crusher Machine rock crushing process into various aggregates better way of running stone crusher crusher plant a flow diagram describing the process of mining coal silica mining technology production line.The invention discloses a production process of battery-grade lithium hydroxide, which comprises the following steps A. ball milling crushing and ball-milling lithium carbonate ore to obtain powder with the granularity of 4-70 um B. dissolution reaction b, adding water into the powder obtained in the step A to prepare lithium carbonate slurry, and adding hydrated lime to react.Patent Li Ne Crusher . Dec 13, 2017 A bearing for a crusher, in particular for a gyratory crusher or cone crusher, may include a housing, an eccentric bush that is mounted in the housing such that the eccentric bush can be rotated about an axle, a bushing that is disposed between the housing and the eccentric bush, and a lubricant line for supplying the bushing with lubricant that.
Calcium carbonate is basically a sedimentary rock that is made of minerals such as calcite or aragonite. Ground Calcium Carbonate is most commonly referred to as GCC, is one of the widely used material in industrial applications. The three basic primary attributes ie. particle size, colour and chemical purity define the quality of the Ground Calcium Carbonate and define.Jul 28 2020 Introduction of limestone powder grinding To engage in the limestone powder grinding industry the most important thing to understand is how the limestone is ground into the powder Limestone powder calcium carbonate ground into different fineness can be roughly divided into two types light calcium carbonate and heavy calcium carbonate.Products Grinding Plant Cone Crusher,crushing. 10tph Silica powder grinding line in Mexico. 30tph Calcium Carbonate powder grinding line in India. 10tph Zeolite powder grinding line in Belgium. 10tph Limestone powder grinding line in Malaysia. 10tph Clinker powder grinding line in Dalian, .'Jaikar' Manufacturer, Supplier and Exporter of.


That little Zombieland cutie Abigail Breslin is growing up quickly. “How quickly?” you ask? Breslin turns seventeen next month! She’s also quite the horror fan.
Not only is she playing a more mature role than we’ve ever seen her in before in this Friday’s serial-killer/chase film The Call (opposite Halle Berry, with director Brad Anderson), but she’s got two more genre projects lined up – Haunter (read more about that here) and Final Girl. Breslin told me a little about each while promoting the call last week in LA.
“Haunter is gonna be at SXSW. It’s directed by Vincenzo Natale, and it’s pretty creepy. So I’m excited for that,” Breslin – an avid fan of ShockTillYouDrop.com – said.
“And I’m such a horror movie fan. It’s my favorite genre. So to be filming it I always have those geek-outs on set like, ‘Oh my God, that’s such a cool shot! I love that!’ And then Final Girl is a movie directed by [photographer and video director] Tyler Shields. It’s not super horror, but it is horrifying in some ways. But it’s a different… The whole experience was so bizarre and strange, so I can’t even fully comprehend it yet. But it’s very different, very different.”
Check out the trailer for Final Girl right here.