Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds

Summary Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study, I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32–34 s, final temperature between 40° C and 47° C.     

Fixation with even small quantities of glutaraldehyde affects red blood cell surface properties in a cell- and species-dependent manner. Studies by cell partitioning

Partitioning in charge-sensitive dextran-poly(ethylene glycol) aqueous phase systems reveals that fixation with even small concentrations of glutaraldehyde (e.g., 0.1% w/v) changes the surface properties of cells. While fixation with larger concentrations of glutaraldehyde (i.e., 1.85%) increases erythrocyte partition ratios, the effect of lower glutaraldehyde concentrations on the partition ratios appears to be species-specific. The differential effect of glutaraldehyde on rat reticulocytes and erythrocytes indicates that fixation is also cell-dependent. These data, together with the previous report that glutaraldehyde fixation does not change the characteristicrelative partition ratios of rat mature erythrocytes of different cell ages, suggest that the nature and extent of glutaraldehyde alteration of cell surfaces must, in each case, be empirically evaluated.     

Comparison of Atomic Force Microscopy Interaction Forces between Bacteria and Silicon Nitride Substrata for Three Commonly Used Immobilization Methods

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.     

Parathyroid cell variants may be provoked during immersion fixation

Summary Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO₄ in the same buffers or, alternatively, in s-collidine.Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5–2% formaldehyde and 2.5–5% acrolein in 0.1 M Na-cacodylate with or without Ca²⁺ and Mg²⁺, Na-phosphate or Na/K-phosphate at 4°C followed by postfixation with 1% OsO₄ in the same buffers or in s-collidine containing sucrose, Ca²⁺ and Mg²⁺. This procedure largely abolished the occurence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Comparison of atomic force microscopy interaction forces between bacteria and silicon nitride substrata for three commonly used immobilization methods

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.     

Some histochemical reactions of mast cells of gerbils,hogs, armadillos and cats

Summary Mast cells of the Mongolian gerbil Meriones unguiculatus, the hog Sus scrofa, the cat Felis catus and the armadillo Pasypus novemcinctus were studied histochemically in relation to various fixation procedures, using azure A at pH 1 and 3, alcian blue at pH 1 and 2.5, diazosafranin at pH 3 and 7.8–8, and the PAS reaction. Fixations were performed in buffered 10% formol and 5% glutaraldehyde, in Kose's fluid, buffered sublimate (B4), lead nitrate and lead acetate formol.With azure A and alcian blue many mast cells were found in the gerbil with the aldehyde fixatives, fewer with the heavy metals. The diazosafranin reaction was present only in the aldehyde material, the PAS reaction was negative.In the hog, mast cells were more numerous after heavy metal fixation, fewer with aldehydes. Azure A stained metachromatically at pH 1 and 3, alcian blue reacted only at pH 1, the PAS reaction was negative, the pH 3 and 8 diazosafranin reactions were positive with all 4 fixations.In the cat, mast cells were moderately numerous with lead acetate formol, rare with formol and absent with glutaraldehyde. They stained with azure A at pH 1 and 3, with alcian blue at pH 1 and 2.5, with diazosafranin at pH 3 and 8 and by the PAS reaction.Armadillo mast cells were more numerous after heavy metal fixations, stained with azure A and alcian blue at pH 1 and 2.5 to 3, and with acid and alkaline diazosafranin.The mast cells of the 4 species vary in their requirements for aldehyde and heavy metal fixation, in their PAS reactivity and in their pH 2.5 alcian blue staining. All are sufficiently sulfated to react to cationic dyes at pH 1, but vary in PAS reactivity, indicating partial or complete sulfation. The presence of 5-hydroxytryptamine is indicated in all four species.Assisted by grant from National Cancer Institute C-04816.     

Artifical rearrangements of insect ooplasm caused by fixation,and their microkymographic recording

Summary Time-lapse photomicrographs of eggs from the Ichneumonid waspPimpla and from the gall midgeWachtliella reveal that during fixation the ooplasm performs excessive streaming movements when conventional fluids such as Bouin's or Dubosq-Brasil's are used. InWachtliella, intravitelline cleavage may continue for two mitotic cycles during fixation. The cytoplasm of individual egg regions may become displaced up to 45% of the egg's length inPimpla, and up to 14% of the egg's length inWachtliella. Therefore fixed preparations may grossly misrepresent the distribution of egg components in the living state. The changes can be traced back by microkymographic registration of movements during fixation. Microkymograms for this purpose were obtained by projecting a time-lapse film through a slit onto photosensitive material mounted on a rotating drum.The shifting of egg components and the mitotic activity of nuclei fail to occur when the egg is treated with a buffered solution of OsO₄ (1%, 1 min). Subsequent fixation with glutaraldehyde results in satisfactory preservation of microtubules.     

Routinely frozen biopsies of human skeletal muscle are suitable for morphological and immunocytochemical analyses at transmission electron microscopy

The aim of the present investigation was to evaluate whether routinely frozen biopsies of human skeletal muscle may be suitable for morphological and immunocytochemical analyses at transmission electron microscopy. The fixation/embedding protocols we successfully used for decades to process fresh mammalian tissues have been applied to frozen muscle biopsies stored for one to four years in liquid nitrogen. After 2.5% glutaraldehyde -2% paraformaldehyde - 1% OsO₄ fixation and embedding in epoxy resin, the ultrastructural morphology of myofibres and satellite cells as well as of their organelles and inclusions proved to be well preserved. As expected, after 4% paraformaldehyde - 0.5% glutaraldehyde fixation and embedding in LR White resin, the morphology of membrane-bounded organelles was relatively poor, although myofibrillar and sarcomeric organization was still recognizable. On the contrary, the myonuclei were excellently preserved and, after conventional staining with uranyl acetate, showed an EDTA-like effect, i.e. the bleaching of condensed chromatin, which allows the visualization of RNP-containing structures. These samples proved to be suitable for immunocytochemical analyses of both cytoskeletal and nuclear components, whereas the poor mitochondrial preservation makes unreliable any in situ investigation on these organelles.Keeping in mind the limitations found, these results open promising perspectives in the study of frozen skeletal muscle samples stored in the tissue banks; this would be especially interesting for rare muscle diseases, where the limited number of biopsies suitable for ultrastructural investigation has so far represented a great restriction in elucidating the cellular mechanisms responsible for the pathological phenotype.Key words: frozen biopsy, electron microscopy, fixation, immunocytochemistry, skeletal muscle.     

Scanning Electron Microscopy of the Influence of Fixation Vehicle Osmolality on Cell Morphology of Spiroplasma, Acholeplasma, and Mycoplasma spp. grown on Agar

Young Spiroplasma citri, corn stunt spiroplasma, and honey bee spiroplasma colonies fixed in 5% glutaraldehyde in M 199 cell culture medium with 0.25 M sucrose showed elongated mycelium-like cells which were sometimes branched or helical. In older colonies beaded chains and rounded bodies were formed. Fixation in 6 % glutaraldehyde in distilled water resulted in amorphous masses in which rounded bodies were present. The spiroplasma cells did not remain osmotically active after glutaraldehyde fixation. Acholeplasma laidlawii and Mycoplasma hyorhinis colonies fixed in glutaraldehyde with or without M 199 medium with 0.25 M sucrose showed little difference in cell morphology.     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.     

Effect of acetone solution in a topical application method on mortality of rice planthoppers, Nilaparvata lugens, Sogatella furcifera, and Laodelphax striatellus (Homoptera: Delphacidae)

The effect of acetone solution on the mortality of rice planthoppers, Nilaparvata lugens (St?l), Sogatella furcifera (Horváth), and Laodelphax striatellus (Fallén), was examined, as it is widely used as a solvent solution in a topical application method for monitoring insecticide susceptibility. The mortality of N. lugens and S. furcifera was significantly higher at 0.32 and 0.28 μl or more acetone droplets per insect than in the control treatment (without acetone), respectively. The mortality of L. striatellus was significantly higher at 0.24 μl or more acetone droplets per insect than in the control treatment. Another commonly used solvent solution, methanol, had a similar effect on L. striatellus. The most standard topical application method uses a hand-operated micro-applicator (Burkard Manufacturing Co., Ltd., UK) equipped with a 50-μl micro-syringe, which can deliver a very small amount of droplets of insecticide/acetone solution per insect, 0.08 μl, so there is no effect of acetone solution on the mortality of N. lugens, S. furcifera, and L. striatellus. However, other equipment for topical application, a repeating dispenser (Hamilton Co., USA) attached with a 10-μl micro-syringe, can deliver a 0.24-μl droplet at a minimum. This equipment can be used for N. lugens and S. furcifera, but not for L. striatellus. It is necessary to choose appropriate equipment for each rice planthopper so that they are not affected by the solvent solution.     

EFFECTS OF FIXATIVES ON THE ULTRASTRUCTURE OF PHYSODES IN VEGETATIVE CELLS OF SCYTOSIPHON LOMENTARIA (SCYTOSIPHONACEAE,PHAEOPHYTA)1

The effects of different glutaraldehyde-osmium fixation schedules on the ultrastructure of the vegetative cells from the meristematic regions of Scytosiphon lomentaria (Lyngbye) Link fronds are described. The best overall preservation of cell structure was obtained with a 2 h fixation in 2.5–3.5% glutaraldehyde in 0.1 M cacodylate buffered seawater (pH 7.0), followed after washing by 1 h post fixation in 1% osmium tetroxide. The addition of 1% caffeine to the glutaraldehyde fixative resulted in better retention and spatial localization of the electron dense phenolic deposits within the cells. Particular attention was paid to the effects of the various fixation schedules on the electron-dense material within the cells and the images obtained were compared with previous accounts of brown algal cells. It is proposed that the term physode should be restricted to the discrete electron dense spherical bodies within the vacuoles and not applied to electron dense material in general. Although the organization of Scytosiphon cells was similar to that previously reported in the Scytosiphonaceae, the organization of the plasmodesmata into pit fields is at variance with previous accounts.     

Phosphotungstic Acid-Chromic Acid as a Selective Electron-Dense Stain for Plasma Membranes of Plant Cells

A mixture consisting of 1% phosphotungstic acid (PTA) in 10% chromic acid (CrO₃) selectively stains the plasma membrane of plant cells. Whole tissue or pelleted cell fractions are prepared for electron microscopy using conventional methods including glutaraldehyde fixation and OsO₄ postfixation, dehydration in acetone and embedding in Epon. To stain the plasma membrane, thin sections are transferred with a plastic loop to the surface of a 1% aqueous solution of periodic acid for 30 min for destaining. Following transfer through 5 distilled water rinses, the sections are exposed to the PTA-CrO₃ mixture for 5 min, rinsed and mounted on grids for viewing with the electron microscope. The selectivity of the stain is retained in homogenates and serves to identify the plant plasma membrane in cell fractions.     

The structure of the ventricle of Elliptio complanatus, a fresh-water lamellibranch

The morphology of the ventricle of the fresh-water lamellibranch, Elliptio complanatus, was investigated. Contrary to the condition reported previously in Tritogonia verrucosa, the two atria in Elliptio communicate with the ventricular lumen through separate openings, each guarded by an atrio-ventricular valve. Fixation of ventricle for electron microscopy with 2.5% buffered glutaraldehyde did not appear to shrink the tissue, in spite of the low blood osmolarity to which the muscle is adapted. Ventricle tissue is composed of smooth muscle fibers, containing a central nucleus, glycogen, mitochondria, paramyosin, dense bodies and “attachment plaques,” much like the ventricle of the salt-water clam, Venus (Mercenaria) mercenaria.     

Fixation of Vervet Monkey Oral Mucosa for Ultrastructural Investigation–Tem

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecui pygerythrta) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolalities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

Rapid detection of food- and waterborne bacteria using surface-enhanced Raman spectroscopy coupled with silver nanosubstrates

Development of rapid and sensitive methods to detect pathogens is important to food and water safety. This study aimed to detect and discriminate important food- and waterborne bacteria (i.e., Escherichia coli O157:H7, Staphylococcus epidermidis, Listeria monocytogenes, and Enterococcus faecelis) by surface-enhanced Raman spectroscopy (SERS) coupled with intracellular nanosilver as SERS substrates. An in vivo molecular probing using intracellular nanosilver for the preparation of bacterial samples was established and assessed. Satisfactory SERS performance and characteristic SERS spectra were obtained from different bacterial samples. Distinctive differences were observed in SERS spectral data, specifically in the Raman shift region of 500–1,800 cm⁻¹, and between bacterial samples at the species and strain levels. The detection limit of SERS coupled with in vivo molecular probing using silver nanosubstrates could reach the level of single cells. Experiments with a mixture of E. coli O157:H7 and S. epidermidis for SERS measurement demonstrate that SERS could be used for classification of mixed bacterial samples. Transmission electron microscopy was used to characterize changes of morphology and cellular composition of bacterial cells after treatment of intracellular nanosilver. The results indicate that SERS coupled with intracellular silver nanosubstrates is a promising method for detection and characterization of food- and waterborne pathogenic and non-pathogenic bacterial samples.     

Gallic acid‐based alkyl esters synthesis in a water‐free system by celite‐bound lipase of Bacillus licheniformis SCD11501

Gallic acid (3, 4, 5‐ trihydroxybenzoic acid) is an important antioxidant, anti‐inflammatory, and radical scavenging agent. In the present study, a purified thermo‐tolerant extra‐cellular lipase of Bacillus licheniformis SCD11501 was successfully immobilized by adsorption on Celite 545 gel matrix followed by treatment with a cross‐linking agent, glutaraldehyde. The celite‐bound lipase treated with glutaraldehyde showed 94.8% binding/retention of enzyme activity (36 U/g; specific activity 16.8 U/g matrix; relative increase in enzyme activity 64.7%) while untreated matrix resulted in 88.1% binding/retention (28.0 U/g matrix; specific activity 8.5 U/g matrix) of lipase. The celite‐bound lipase was successfully used to synthesis methyl gallate (58.2%), ethyl gallate (66.9%), n‐propyl gallate (72.1%), and n‐butyl gallate (63.8%) at 55^oC in 10 h under shaking (150 g) in a water‐free system by sequentially optimizing various reaction parameters. The low conversion of more polar alcohols such as methanol and ethanol into their respective gallate esters might be due to the ability of these alcohols to severely remove water from the protein hydration shell, leading to enzyme inactivation. Molecular sieves added to the reaction mixture resulted in enhanced yield of the alkyl ester(s). The characterization of synthesised esters was done through fourier transform infrared (FTIR) spectroscopy and ¹H NMR spectrum analysis. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:715–723, 2015     

Rapid microwave fixation--a comparative morphometric study

Summary Tissue blocks taken from healthy human lung tisues, from primary bronchial carcinoma and from mediastinal and hilar lymph nodes were placed in the following solutions. Tris buffer; buffered formalin (0.5%, 1%, 7%); 0.1 mol NaCl; distilled water; DMSO (1%, 10%, 20%); acetone (10%); methanol (50%, 80%, 100%); glutaraldehyde (2.5%), and fixed by use of a commercial microwave oven.Tissue blocks obtained from the same surgical specimens were fixed in 7% buffered formalin for 24 h for comparison. Conventional and microwave-fixed tissue was embedded in paraffin, and stained with haematoxylin and eosin. Fifteen specimens of each group and each solution were examined by light microscopy. Minimum diameter and area of 100 nuclei of each specimen were measured interactively.Histomorphological sections fixed with Tris buffer in a microwave oven revealed best morphological results showing more contrast in chromatin distribution of nuclei and opening of interstitial lung capillaries in comparison to conventional formalin-fixed specimens. No statistically significant differences in area and minimum diameter of nuclei between the different groups were found. Microwave fixation using Tris buffer is a time-saving fixation method at least comparable to conventional formalin fixation. It is not accompanied by hazards to the environment that are unavoidable by formalin fixation.     

Optimization of Two Immunofluorescent Antibodies for the Detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> Using Immunofluorescent Microscopy and Flow Cytometry

Two commercially available fluorescein isothiocyanate (FITC) -conjugated anti-Escherichia coli antibodies, tested for immunofluorescence were assessed for their suitability in screening E. coli using flow cytometry. Staining efficacy was initially tested using immunofluorescent microscopy; and further optimization was carried out using flow cytometry. Initially, an acetone fixation step was utilized; however, it was determined statistically that the step could be omitted without impacting the assay and thus reduce the time involved. There was no statistical difference between the staining proficiency of the two antibodies employed. The percentage staining was quite low, approximately 10% for the two antibodies, which indicated that both were equally sensitive but ultimately, more specific antibodies are required for the detection of E. coli. Known proportions of target-E. coli (10⁵, 10⁶, and 10⁷ cells/ml) were mixed with large quantities of non-target bacteria; there was a significant correlation among all the antibodies at the different bacterial cell concentrations. Therefore, despite the low staining percentage achieved on the bacterial cultures, there is a representative and comparative level of staining occurring, between samples and between bacterial strains.     

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol‐butanol‐ethanol fuel mixture

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone‐butanol‐ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol‐butanol‐ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab‐scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot‐scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab‐scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1083–1088, 2013