Embedding Agent Research Articles

Micromilling vs hand drilling in stable isotope analyses of incremental carbonates: The potential for δ13 C contamination by embedding resin.

RationaleEmbedding resins are commonly used to facilitate high‐resolution sampling for stable isotope analysis but anomalous δ13C values have been observed in some cases. Here we compare the results of microsampling strategies for hand‐drilled versus resin‐embedded micromilled samples from the same marine shells to assess whether resin contamination is implicated in δ13C spikes. The comparison allows assessment of the relative benefits for spatial resolution, seasonal range for both δ18O and δ13C, and sample failure rates.MethodsHand‐drilled samples were obtained from two bivalve shells (Spisula sachalinensis), corresponding to micromilled samples on the same shells where high δ13C spikes were observed. All carbonate powders were analysed using a dual‐inlet Isoprime mass spectrometer and Multiprep device. Results from both sample sets were compared statistically.ResultsNo anomalous high δ13C values and no failures due to insufficient gas were observed in the hand‐drilled samples in contrast to the embedded micromilled sequences. Spatial resolution was reduced (~2.5×) in the former compared with the latter, resulting in a small reduction in the total range observed in the micromilled δ13C and δ18O values. Reduced sampling resolution between the two datasets was only significant for δ18O.ConclusionsFor S. sachalinensis (as with other similar bivalves), rapid growth mitigates the reduced sampling resolution of hand drilling and does not significantly impact observed isotopic range and seasonal patterning. Occurrence of anomalous δ13C values were eliminated and failure rates due to insufficient sample size greatly reduced in the hand‐drilled dataset. We can find no other explanation for the occurrence of δ13C spikes than contamination by the embedding agent. We conclude that the logistical and interpretational benefits of careful hand drilling may be preferable to resin embedding for micromilling in marine shells, corals or speleothems where growth rate is rapid and the highest resolution is not required.

Sample preparation optimization of insects and zebrafish for whole-body mass spectrometry imaging.

Appropriate sample preparation is one of the most critical steps in mass spectrometry imaging (MSI), which is closely associated with reproducible and reliable images. Despite that model insects and organisms have been widely used in various research fields, including toxicology, drug discovery, disease models, and neurobiology, a systematic investigation on sample preparation optimization for MSI analysis has been relatively rare. Unlike mammalian tissues with satisfactory homogeneity, freezing sectioning of the whole body of insects is still challenging because some insect tissues are hard on the outside and soft on the inside, especially for some small and fragile insects. Herein, we systematically investigated the sample preparation conditions of various insects and model organisms, including honeybees (Apis cerana), oriental fruit flies (Bactrocera dorsalis), zebrafish (Danio rerio), fall armyworms (Spodoptera frugiperda), and diamondback moths (Plutella xylostella), for MSI. Three cutting temperatures, four embedding agents, and seven thicknesses were comprehensively investigated to achieve optimal sample preparation protocols for MSI analysis. The results presented herein indicated that the optimal cutting temperature and embedding agent were -20°C and gelatin, respectively, providing better tissue integrity and less mass spectral interference. However, the optimal thickness for different organisms can vary with each individual. Using this optimized protocol, we exploited the potential of MSI for visualizing the tissue-specific distribution of endogenous lipids in four insects and zebrafish. Taken together, this work provides guidelines for the optimized sample preparation of insects and model organisms, facilitating the expansion of the potential of MSI in the life sciences and environmental sciences.

Performance Comparison of Two Cryosection Embedding Agents Used for Desorption Electrospray Ionization Mass Spectrometry Imaging

To evaluate the potential effect of embedding with carboxylmethyl cellulose (CMC) and embedding with optimal cutting temperature (OCT) compound followed by washing with PBS (OCT-W) on the analysis of breast cancer tissue samples with desorption electrospray ionization mass spectrometry imaging (DESI-MSI). DESI-MSI of fresh frozen (FF) tissue samples, OCT-embedded samples, CMC-embedded samples, and OCT-W samples from the same breast cancer tumor tissue were performed. The ratio of maximum abundance ion was used to assess the reproducibility of DESI-MSI analysis. In addition, the effects of the treatment of each group were examined by comparing the characteristic ion species and the ion signal intensity detected by DESI-MSI. DESI-MSI of continuous sections of FF samples showed that the coefficient of variation (CV) of the pair-to-pair ratios of m/ z 281.25, m/ z 309.28 and m/ z 279.23 ions, the three ions with the highest intensity in the tumor region, were 19.61%, 20.74% and 10.18%, respectively. The characteristic ion species detected by DESI-MSI of CMC embedded tissue and the OCT-W tissue were almost the same, compared with those of the FF tumor tissue. However, ion species detected in OCT embedded samples were less than 50% of the FF samples. In terms of ion signal intensity, the CMC embedded tissue was not affected overall, while the signal of most of the characteristic ions of the OCT-W group showed decreased intensity (P<0.05). FF tissue sections and CMC-embedded samples can be used for DESI-MSI routine analysis. OCT embedding affects the feasibility of sample analysis whether or not the sample undergoes washing with PBS. CMC embedding agent is recommended if the tissue sections need to be fixated and supported due to small sample size, fragility, or other problems.

Remediation of chromium- and fluoride-contaminated groundwater by immobilized Citrobacter sp. on a nano-ZrO2 hybrid material.

To effectively address excessive SO42-, Cr(VI), total chromium and F- in the groundwater of acidic mining areas, a facultative anaerobic bacterium, Citrobacter, with sulfate-reducing properties, tolerance to hexavalent chromium and the ability to reduce Cr(VI) to Cr(III) was isolated and domesticated. Based on microbial immobilization technology, a nano-ZrO2 polyacrylamide hybrid material was prepared as an embedding agent to form nano-ZrO2 polyacrylamide Citrobacter (ZPC) particles. ZPC was microscopically characterized, and the removal performance and mechanism of ZPC for SO42-, Cr(VI), total chromium and F- in groundwater were analyzed. The results of single-factor tests showed that the optimal reaction conditions included a reaction temperature of 35°C, Citrobacter dosage of 35% (volume ratio) in the particles and hybrid material dosage of 300 mL; under these conditions, the removal rates of SO42-, Cr(VI), total chromium and F- were 70.5%, 100%, 100% and 93.3%, respectively, and the pH value increased from 4.6 to 8.07. On this basis, the effects of the reaction layer type, influent hydraulic load and influent concentration on the removal efficiency of polluted groundwater were studied through dynamic experiments. The experimental results showed that ZPC particles were better than Citrobacter as a reaction layer; the optimal influent hydraulic load was 3.0 m3/(m2·d); the selectivity of ZPC particles to anions and anionic groups was different; and the order of adsorption selectivity was F- > Cr(VI) > SO42-.

Effective immobilization of Bacillus subtilis in chitosan-sodium alginate composite carrier for ammonia removal from anaerobically digested swine wastewater

To overcome the easy loss of microorganism, the mass production of sludge and the consumption of aeration energy during biological treatment of anaerobically digested swine wastewater, this study used chitosan–sodium alginate composite carrier to prepare immobilized bacteria pellets. The heterotrophic bacteria tolerant to high concentrations of ammonia nitrogen were isolated and the conditions for immobilizing bacteria were optimized. The performance of immobilized bacteria pellets to remove ammonia nitrogen from ADSW was determined and the corresponding mechanism was investigated. Results showed that the isolated bacteria were Bacillus subtilis, and the optimal conditions to prepare the immobilized bacteria pellets by response surface methodology tests were sodium alginate of 0.84% (m/V), chitosan of 0.22% (m/V), embedding time of 32 min and embedding amount of 15% (V/V). In ADSW treatment, at pH 6, 20 g/L of the immobilized bacteria pellets removed 96.5% of ammonia nitrogen. Both adsorption and microbial action contributed to ammonia nitrogen removal, and their contributions were 54.3% and 42.2%, respectively. Compared with the immobilized bacteria pellets using chitosan–sodium alginate as carrier, the one using mono alginate as carrier had a weaker ability to remove ammonia nitrogen, with a removal efficiency of 67.4%. The main mechanism was the formation of polyelectrolyte membrane by the connection between amino groups of chitosan and carboxyl groups of sodium alginate, which stabilized the immobilized bacteria pellets and prolonged their service life. To sum up, the immobilized bacteria pellets using chitosan–sodium alginate as an embedding agent have a promising prospect in ammonia nitrogen removal from wastewater.

Permeabilization and immobilization of whole-cell Pseudomonas nitroreducens SP.001 to improve its l-glutaminase performance.

L-Glutaminase is considered to be an important industrial enzyme in both the pharmaceutical and food industries, especially for producing functional glutamyl compounds, such as l-theanine. Pseudomonas nitroreducens SP.001 with intracellular l-glutaminase activity has been screened previously. In the present study, three physical permeabilization methods were used to improve l-glutaminase activity. Then, the whole-cell immobilization conditions of permeabilized cells using sodium alginate as an embedding agent were optimized to enhance the enzyme's stability and reusability. The characteristics of the immobilized cells were investigated in comparison with those of permeabilized cells. The results obtained showed that cell permeabilization using osmotic shock with 155 g L-1 sucrose markedly improved enzyme activity. Then, an effective procedure for immobilization of permeabilized P. nitroreducens cells was established. The optimum conditions for cell immobilization were: sodium alginate 40 g L-1 , calcium chloride 30 g L-1 , cell mass 100 g L-1 and a curing time of 3 h. After successful immobilization, characterization studies revealed that the thermostability and pH resistance of l-glutaminase from immobilized cells were enhanced compared to those from permeabilized cells. Moreover, the immobilized biocatalyst could be reused up to 10 times and retained 80% of its activity. The stability and reusability of the permeabilized cells were improved through the immobilization. These findings indicated that immobilized whole-cell l-glutaminase from P. nitroreducens SP.001 possesses more potential for various industrial biotechnological applications than free cells. © 2020 Society of Chemical Industry.

Novel Method for Preparing Transmission Electron Microscopy Samples of Micrometer-Sized Powder Particles by Using Focused Ion Beam.

The preparation of transmission electron microscopy (TEM) samples from powders is quite difficult and challenging. For powders with particles in the 1-5 μm size range, it is especially difficult to select an adequate sample preparation technique. Epoxy is commonly used to bind powder, but drawbacks, such as differential milling originating from unequal milling rates between the epoxy and powder, remain. We propose a new, simple method for preparing TEM samples. This method is especially useful for powders with particles in the 1-5 μm size range that are vulnerable to oxidation. The method uses solder as an embedding agent together with focused ion beam (FIB) milling. The powder was embedded in low-temperature solder using a conventional hot-mounting instrument. Subsequently, FIB was used to fabricate thin TEM samples via the lift-out technique. The solder proved to be more effective than epoxy in producing thin TEM samples with large areas. The problem of differential milling was mitigated, and the solder binder was more stable than epoxy under an electron beam. This methodology can be applied for preparing TEM samples from various powders that are either vulnerable to oxidation or composed of high atomic number elements.

Efficient sensory-grounded grasp pose quality mapping for gripper design and online grasp planning

The gentle grasping and manipulation of objects in dense un-structured environments, such as the agricultural, food processing, or home environments constitute a formidable challenge for robotic systems. Knowledge regarding wrist poses (wrist positions and orientations) that may lead to successful grasps is especially important in such environments for both gripper design and online grasp planning. Graspability maps store grasp quality grades at different wrist poses in object-centered coordinates. Previously graspability maps were derived based on object models in a lengthy, offline process and thus had limited usability. We have developed geometry-based grasp quality measures related to classical grasp quality measures, which can be determined directly from a 3D point cloud. This facilitates embedding agent perception capabilities within the grasp quality determination. Additionally by scanning the object’s surface for finger contact points rather than scanning the volume of the bounding box about the object, and by using parallel computation, graspability map computation-time is considerably reduced, facilitating online computation of multiple measures. We validate the developed measures in a physical environment, show that computation-time can be reduced by more than 90% with very low reduction in map quality, and show the applicability of the developed methods for both simple and complex objects.

The Preparation and Research of PVA Embedding Zr-AC Phosphorus Adsorbent

The hydration ZrOH was loaded in the powdered activated carbon（PAC）,the aim to modify activated carbon, then the mixed liquor including poval(PVA) and sodium alginate tech grade (SV) , as the embedding agent ,embedded the modified PAC.The main investigation were that the hydration ZrOH concentration,the ratio of embedding agent composition and Curing liquid ingredient etc. influence the phosphorus removal . The orthogonal experiment determined the optimum prepared conditions of embedding adsorbent. The experimental results showed that : the optimum loaded conditions were that PAC average particle size was 0.5-1.0mm, hydration ZrOH concentration was 0.04mol/L;the optimum embedded preparation conditions were PVA mass concentration for 8%，SV mass concentration for 1.2%; the optimum cured conditions were the curing liquid ingredient was saturated boric acid with 2% CaCl2. The adsorption of phosphorus removal efficiency was 99.27%.

Optimization of Entrapping Conditions of Nitrifying Bacteria and Selection of Entrapping Agent

Sodium alginate and polyvinyl alcohol were selected as entrapping agents to immobilize nitrifying microbia. Through orthogonal tests, optimal conditions of entrapping nitrifying bacteria by the two agents were determined by ammonia nitrogen removal efficiency. The properties of the two immobilized microbial beads produced under their optimum embedding conditions were compared. Results indicate that the optimum embedding conditions of calcium alginate beads (briefly CA beads) were 10ml of nitrifying bacteria, 4% of sodium alginate content, 3mm in bead diameter and 24-hr crosslinking time. The optimum embedding conditions of polyvinyl alcohol beads (briefly PVA beads) were 10ml of nitrifying bacteria, 10% of PVA content, 3mm in bead diameter and 24-hr crosslinking time. Comprehensive comparisons on mechanical strength, stability, mass transfer property and ammonia nitrogen removal efficiency of the two immobilized microbial beads indicate that polyvinyl alcohol is a better embedding agent for nitrifying bacteria in wastewater treatment compared with sodium alginate.

Examination of the Penetration of Polymeric Methylene Di-Phenyl-Di-Isocyanate (pMDI) into Wood Structure Using Chemical-State X-Ray Microscopy

Summary The penetration behaviour of isocyanate-based wood resins was evaluated using x-ray microscopy. Aspen wood pieces were bonded together in a controlled manner. These were embedded in a methacrylate-based resinand thin sections were prepared, cut from the transverse face of thewoodcomposite. X-ray images of these sections were prepared at several selected x-ray energies to allow the isocyanate, cellulose, lignin and the embedding agent distributions to be mapped. The isocyanate resin was found to penetrate deeply into the wood. The resin enters large cell lumen, and wicks along the inner cell wall surfaces. The resin accesses connected cells via connecting pits, which become filled with the resin. The affinity of the isocyanate to the inner surfaces of the large cells is probably due to the hydrophobicity of these surfaces. Isocyanate resins do not penetrate into the smaller parenchyma and tracheid cells and indeed do not even wet the inner surfaces of these cells where isocyanate entry has been allowed due to damage of the cell at the macroscopic surface of the wood. If isocyanates penetrate into the wood-cell walls of the large cells, the concentration in the cell walls has been determined to be less than 2%of the bulk concentration. This lower limit is the sensitivity limit imposed by photon statistics in the data.

CP embedding agent and its application to the serial section of the body wall tissue of insects

The key of tissue section technique is the selection and application of the embedding agent. Based on the specific characteristics of the tissue and outer skeleton of insects, a new kind of embedding agent, colophony and paraffin (CP), was used without destroying the integrity of insect organs. The tissue can be sliced continuously by the use of CP embedding technique. It solves the 3 difficult problems in the serial section of the organ and tissue of insect, namely stiff materials, thin section and continuity of sections. These difficult problems are not solved by any unitary tissue section technique that is used now, including the paraffin section technique. By using the CP embedding technique, any parts or organs (like antenna, leg and wing) and even the whole body of insects (mini body) can be sliced up continuously. It will open up wide possibilities for the study of inner structure, the interrelations among organs, the micro-characteristics and the physiological functions of insects systematically.

Methodology for determining the degree of impregnation from continuous glass fibre prepreg

A procedure to determine the degree of impregnation from melt-impregnated polypropylene/glass fibre prepreg has been developed. The prepeg sample is moulded in an embedding agent, ground and coloured with colouring agent penetrating to the space between unimpregnated fibres. The sample is studied with an optical microscope and transillumination is used. Unimpregnated fibres are calculated individually. Moulding the specimens with embedding agent, grinding and preparing the specimens with colouring agent, postgrinding, study of the specimens and the calculations to determine the degree of impregnation are described.

LR White acrylic resin polymerized by microwave energy for immuno-EM ofSaccharomyces cerevisiae: A rapid method

Microwave embedment methods have been developed for epoxy and polyester resins. Because the thick yeast cell wall is known to inhibit penetration by many embedding media, we wished to use an embedding agent exhibiting favorable properties of hydrophilicity and low viscosity. The complex requirement for low temperature and curing using UV irradiation with the polymer Lowicryl, and recent advantages described for yeast using LR White, made the latter polymer a good candidate for our studies.Saccharomyces cerevisiaewas grown at 30°C in selective medium to an OD640= 1.0. A modification of the procedure of Tuinen and Riezman was used to fix the cells. Briefly, the cells were washed three times with Sorenson's buffer, pH 7.3, and fixed as a suspension in 3% paraformaldehyde, 0.25% glutaraldehyde in Sorenson's for 2 hours at room temperature. The cells were again washed three times followed by incubation in 1 % sodium meta-periodate at room temperature for one hour.

Modifications of a Method for Preparing Retinal Wholemounts for Sectioning

A method for embedding and sectioning retinal wholemounts is described. It employs a hydrophilic embedding agent, LR White, and coverslips inert to standard embedding resins. This simple technique, which has worked successfully on both enzymatically and autoradiographically labelled material, provides a means by which retinal wholemounts can be cut nearly parallel to the plane of the retinal layers. Previously prepared retinal wholemounts can also be successfully embedded and sectioned more than a year later by the method described.

A method of relating craniofacial sections to topography in embryos.

The transparent properties of the embedding agent glycol methacrylate facilitate orientation of the complex embryonic craniofacial region. This technique allows for consistent and reproducible section-to-topography orientation. We find it to be a valuable adjunct for envisioning three-dimensional relationships. The contrast of external features of the embryo is enhanced when stained lightly with hematoxylin prior to embedding. The craniofacial region of the embedded embryo is removed with a fine surgical saw and reimbedded. Section-to-topography relationships are readily monitored and documented photographically. Furthermore, it is possible to "preview" sections for symmetry and other considerations of orientation by viewing the cutting face of the block under oblique illumination. A relief image of structures is then visible.

Ultrastructure of Escherichia coli cells infected with bacteriophage R17.

Franklin, Richard M. (Institut de Recherches sur le Cancer, Villejuif, Seine, France), and Nicole Granboulan. Ultrastructure of Escherichia coli cells infected with bacteriophage R17. J. Bacteriol. 91:834-848. 1966-Ultrastructural changes in Escherichia coli cells infected with ribonucleic acid (RNA) bacteriophage R17 were studied under conditions of one-step growth. No morphological alterations were seen during the latent period. During the period of rapid viral synthesis, a fibrillar lesion surrounded by ribonucleoprotein particles was observed in a polar region. Late in infection, paracrystalline arrays of virions were found in over 90% of the cells. When protein synthesis was blocked by in over 90% of the cells. When protein synthesis was blocked by chloramphenicol at 20 min postinfection, allowing continued viral RNA synthesis without production of coat protein, a dense fibrillar area appeared in a paranuclear region. Cytochemical studies were done on cells embedded in hydroxypropyl methacrylate, a water-miscible embedding agent. The paracrystalline arrays of virions were digested after extensive treatment with either pepsin or ribonuclease. Shorter digestion with the pepsin resulted in better definition of the crystal regions. The fibrillar area found in chloramphenicol-treated cells was digested by ribonuclease but not by pepsin, and was also resistant to lead extraction. This region probably represents a pool of virus-specific RNA.

Carbowax embedding for obtaining thin tissue sections and study of intracellular lipids.

Carbowax, a water soluble wax, as an embedding agent is a valuable adjunct to the armamentarium of the tissue technologist. This report is intended to supplement previous publications on the use of Carbowax and to indicate die necessity for preheating and variation of Carbowax mixtures according to the climate.Carbowax embedding provides an easy means for obtaining tissue sections 1 to 3 μ in thickness either with or without previous exposure to fat solvents. These sections are admirably suited for cytological study, particularly of intracellular lipoids.

A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.The methods for sectioning, and the removal of the embedding agent are given.The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.