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A Formaldehyde-Glutaraldehyde Fixative of High Osmolality for Use in Electron Microscopy

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Morris John Karnovsky at Harvard Medical School
Morris John Karnovsky
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A Formaldehyde-Glutaraldehyde Fixative of High Osmolality for Use in
Electron Microscopy
MORRIS J. KARNOVSKY. Department of Pathology, Harvard Medical School,
Boston, Massachusetts
A combined formaldehyde-glutaraldehyde fixative has yielded excellent fixation
of a wide variety of tissues. 2 gm of paraformaldehyde powder are dissolved in
25 ml water by heating to 60-70°C and stirring. One to three drops of 1 N
NaOH are added with stirring until the solution clears. A slight milkiness may
persist. The solution is cooled, 5 ml of 50 percent glutaraldehyde (Biological
Grade, Union Carbide Company) are added, and the volume is made to 50 ml
with 0.2 M cacodylate or phosphate buffer, pH 7.4-7.6. The final pH is 7.2. If
cacodylate is used, 25 mg CaCl2 anhydrous is added. Slabs of tissue 3 to 4 mm
thick are fixed at room temperature for 20 to 30 minutes and are then diced
into small blocks, and fixation is continued at room temperature for 2 to 5
hours. The blocks are washed for 3 to 12 hours in cold 0.1 M buffer, are
postfixed in cold 1.33 percent osmium tetroxide buffered with
s
-collidine for 2
hours, and are embedded as usual.
It is of interest that the osmolality of this fixative is about 2010 milliosmols per
kg. Despite this high osmolality, shrinkage is unusual except when the fixative
is perfused or applied to free floating cells and monolayers; then dilution seems
desirable (Williams and Gould, work in progress). Tissue fluid in slabs of tissue
probably dilutes the fixative as it penetrates, reducing the osmolality somewhat.
Myelin figures are less commonly seen than with formaldehyde or
glutaraldehyde fixation alone, and all cell components are well preserved
except lipid droplets, which are extracted. Microtubules are particularly well
preserved. It is surmised that the formaldehyde penetrates faster than the
glutaraldehyde and temporarily stabilizes structures which are subsequently
more permanently stabilized by glutaraldehyde.
Supported by grant HE-09125 from the NIH, USPHS. The author is the recipient
of a Lederle Medical Faculty award. .
... Samples were collected in the initial condition and after 30 and 60 days. The samples were fixed in Karnovsky solution (Karnovsky 1965) for 24 h, dehydrated in an ethanol series, and embedded in 2-hydroxyethyl methacrylate according to the protocol described by Paiva et al. (2011). Cross and longitudinal sections (3 µm thick) were obtained using a rotary microtome, stained with 0.05% toluidine blue, pH 7.4 (O'Brien et al. 1964, modified), and mounted on permanent slides using acrylic resin. ...
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Physiological and cytological responses to thermal stress in recalcitrant seeds of Mauritia flexuosa (Arecaceae)
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Introduction Bacillus velezensis is a ubiquitous bacterium with potent antifungal activity and a plant growth promoter. This study investigated the potential of B. velezensis CNPMS-22 as a biocontrol agent against phytopathogenic fungi under diverse experimental conditions and its potential as a plant growth promoter. Genome sequencing and analysis revealed putative genes involved in these traits. Methods This research performed in vitro experiments to evaluate the CNPMS-22 antagonistic activity against 10 phytopathogenic fungi using dual culture in plate (DCP) and inverted sealed plate assay (ISP). Greenhouse and field tests evaluated the ability of CNPMS-22 to control Fusarium verticillioides in maize plants in vivo. The CNPMS-22 genome was sequenced using the Illumina HiSeq 4,000 platform, and genomic analysis also included manual procedures to identify genes of interest accurately. Results CNPMS-22 showed antifungal activity in vitro against all fungi tested, with notable reductions in mycelial growth in both DCP and ISP experiments. In the ISP, volatile organic compounds (VOCs) produced by CNPMS-22 also altered the mycelium coloration of some fungi. Scanning electron microscopy revealed morphological alterations in the hyphae of F. verticillioides in contact with CNPMS-22, including twisted, wrinkled, and ruptured hyphae. Eight cluster candidates for synthesizing non-ribosomal lipopeptides and ribosomal genes for extracellular lytic enzymes, biofilm, VOCs, and other secondary metabolites with antifungal activity and plant growth promoters were identified by genomic analysis. The greenhouse and field experiments showed that seed treatment with CNPMS-22 reduced Fusarium symptoms in plants and increased maize productivity. Conclusion Our findings highlight the CNPMS-22’s potential as bioinoculant for fungal disease control and plant growth with valuable implications for a sustainable crop productivity.
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... After sacrificing P10 WT and Cobl KO mice, the soft tissue of the lower jaw was removed and the remaining bone including the teeth was incubated at 4 • C in Karnovsky's solution [20] until further processing. After 24 h of water immersion, the tissue was dried and subsequently blocked in Epofix (Struers GmbH, Willich, Germany) for another 24 h. ...
The Evolutionary Young Actin Nucleator Cobl Is Important for Proper Amelogenesis
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The actin cytoskeleton plays an important role in morphological changes of ameloblasts during the formation of enamel, which is indispensable for teeth to withstand wear, fracture and caries progression. This study reveals that the actin nucleator Cobl is expressed in ameloblasts of mandibular molars during amelogenesis. Cobl expression was particularly pronounced during the secretory phase of the enamel-forming cells. Cobl colocalized with actin filaments at the cell cortex. Importantly, our analyses show an influence of Cobl on both ameloblast morphology and cytoskeletal organization as well as on enamel composition. At P0, Cobl knock-out causes an increased height of ameloblasts and an increased F-actin content at the apical membrane. During the maturation phase, the F-actin density at the apical membrane was instead significantly reduced when compared to WT mice. At the same time, Cobl-deficient mice showed an increased carbon content of the enamel and an increased enamel surface of mandibular molars. These findings demonstrate a decisive influence of the actin nucleator Cobl on the actin cytoskeleton and the morphology of ameloblasts during amelogenesis. Our work thus expands the understanding of the regulation of the actin cytoskeleton during amelogenesis and helps to further elucidate the complex processes of enamel formation during tooth development.
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... The micromorphology of the gall surface, the oak leaf surface, and the internal structure of the galls were analyzed using the JEOL JSM 6390LV scanning electron microscope, at the Mossakowski Medical Research Centre, Polish Academy of Sciences in Warsaw. Fragments of plant material (2 × 2 mm) were fixed in 2.5% glutaraldehyde for 12 h at 4 • C in 0.1M cacodylate buffer (pH 7.4) [34]. They were then rinsed several times in the same buffer for 12 h. ...
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The structural and ultrastructural characteristics of galls induced by three species of insects parasitizing on oak leaves (Quercus robur L.) were examined utilizing light and fluorescent microscopes, as well as scanning and transmission electron microscopes. The tissues of the investigated galls exhibited marked differences from those of a typical oak leaf. In the Cynips quercusfolii L. gall, the larval chamber in its final stage was formed from the remnants of dead cells that remained after larval feeding on the gall nutritive tissue. The cells of the gall nutritive tissue and the cells of the gall parenchyma exhibited diametrical differences: the former contained dense cytoplasm and had large nuclei and nucleoli, whereas the latter displayed sparse cytoplasm, prominent vacuoles, and very small nuclei. The region of coalescence between the gall stalk and leaf tissues has been described. In Neuroterus numismalis Geoffroy gall, the early developmental phases have been described in detail. The external gall tissues resembled periderm, whereas periderm does not normally occur in leaves. In the cytoplasm of Cynips longiventris Hartig gall, different bodies were found, including organized smooth endoplasmic reticulum; however, not all of the observed structures were definitively classified.
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... The effects of the storage methods over the wall ultrastructure were verified on the hydrated and frozen-desiccated spores stored for 275 days. The hydrated samples had moisture excess removed and were immediately fixed in paraformaldehyde 2% and glutaraldehyde 2.5% solution in 0.2 M phosphate buffer (pH 7.3) (Karnovsky 1965). The frozen samples were recovered, following the unfreezing method described in Section 2.3.1 and then fixed in the same solution. ...
A Simple and Effective Method for the Cryopreservation of Quillwort Spores
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Quillworts are highly threatened worldwide and many Isoëtes species have been suffering population decreases. Isoëtes cangae is a lycophyte endemic from a single Amazon lake that has been challenging due to climate change and deforestation of the Amazon rainforest. The development of strategies for ex situ conservation of lycophyte germplasm is urgent to prevent biodiversity losses. Spores of I. cangae were obtained from specimens collected in situ. Samples were submitted to three different moisture conditions: hydrated, air‐dry desiccation and drying in silica gel. Desiccated samples were stored in an ultrafreezer (−80°C) or liquid nitrogen (−196°C) and the hydrated spores at room temperature. The spore viability was evaluated through spore germination (G) and sporeling emergence (SE) indices after 30, 90, 180 and 275 days of storage. The growth of emerged sporophytes was evaluated after 3 months. Desiccated–cryopreserved spores had high G% and SE%, even after 275 days of storage, and did not differ from hydrated spores. Hydrated spores had higher daily emergence and a longer time to maximum sporophyte emergence than desiccated spores, but not in all storage periods. Sporophytes that emerged from hydrated and desiccated spores had a similar development. Both desiccated methods and temperatures of storage maintained spore viability high. The methodologies tested in this study are novel for the genus and represent a significant step towards the ex situ conservation of quillworts.
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Silicon mitigates the adverse effects of drought on Lolium perenne physiological, morphometric and anatomical characters
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Electron Microscopy of Structural Detail in Frozen Biological Specimens
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Cytochemistry and electron microscopy
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  • Apr 1963
The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.
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