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Satoshi Mitarai - One of the best experts on this subject based on the ideXlab platform.
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Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and freeze-substitution with osmium-acetone at ultralow temperature.
Journal of Microbiological Methods, 2013Co-Authors: Hiroyuki Yamada, Kinuyo Chikamatsu, Akio Aono, Satoshi MitaraiAbstract:Abstract Sample preparations for transmission electron microscopy of virulent Mycobacterium tuberculosis are usually performed with chemical fixation using glutaraldehyde (GA) in a Biosafety area followed by post-fixation with aqueous osmium tetroxide (OT) in a conventional laboratory outside the Biosafety area. Freeze-substitution with osmium-acetone (OA) at ultralow temperature (− 85 °C) has been shown to provide high quality final images and preserves cellular structures intact. However, some preparation procedures for freeze-substitution often require large fixed devices for freezing in a special laboratory. We have reported a novel freeze-substitution preparation method that can be performed using a portable device in a Biosafety Cabinet at Biosafety level (BSL) 3 areas. Here, as a next step, we examined whether images obtained from rapid freeze-substitution (RFS) after fixation with glutaraldehyde (GA > RFS) are of comparable quality to those obtained using standard RFS. GA > RFS provided excellent preservation of mycobacterial cell ultrastructure, including visualization of cytoplasmic ribosomes, DNA fibers, and the outer membrane. The average number of ribosomes per cubic micrometer counted on RFS and GA > RFS was not significantly different (6987.8 ± 2181.0 and 6888.9 ± 1799.3, respectively). These values were higher, but not significantly so, than those obtained using conventional chemical fixation (5018.7 ± 2511.3). This procedure may be useful for RFS preparation of unculturable mycobacteria strains or virulent strains isolated in laboratories that cannot perform RFS.
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Novel freeze-substitution electron microscopy provides new aspects of virulent Mycobacterium tuberculosis with visualization of the outer membrane and satisfying Biosafety requirements.
Journal of microbiological methods, 2009Co-Authors: Hiroyuki Yamada, Satoshi Mitarai, Kinuyo Chikamatsu, Kazue Mizuno, Masashi YamaguchiAbstract:Transmission electron microscopy (TEM) of virulent bacteria is usually performed following chemical fixation (CF) with aldehyde fixatives such as glutaraldehyde because of the Biosafety problem. However, CF may alter sample ultrastructure. In this study, we used a rapid-freeze substitution (RFS) sandwich method without pre-embedding in agar. TEM images obtained using this method were completely different from those of conventional chemically fixed samples; the bacilli cytoplasm of the RFS preparations was filled evenly with numerous ribosomes, and there was no positional variation of electron density that was obvious in those obtained with CF samples. The sandwich method is suitable for microbiological materials without expensive devices and can be easily performed in a Biosafety Cabinet. In future, this method coupled with novel labeling techniques may help localize structural and functional molecules throughout a bacterial cell.
Hiroyuki Yamada - One of the best experts on this subject based on the ideXlab platform.
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Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and freeze-substitution with osmium-acetone at ultralow temperature.
Journal of Microbiological Methods, 2013Co-Authors: Hiroyuki Yamada, Kinuyo Chikamatsu, Akio Aono, Satoshi MitaraiAbstract:Abstract Sample preparations for transmission electron microscopy of virulent Mycobacterium tuberculosis are usually performed with chemical fixation using glutaraldehyde (GA) in a Biosafety area followed by post-fixation with aqueous osmium tetroxide (OT) in a conventional laboratory outside the Biosafety area. Freeze-substitution with osmium-acetone (OA) at ultralow temperature (− 85 °C) has been shown to provide high quality final images and preserves cellular structures intact. However, some preparation procedures for freeze-substitution often require large fixed devices for freezing in a special laboratory. We have reported a novel freeze-substitution preparation method that can be performed using a portable device in a Biosafety Cabinet at Biosafety level (BSL) 3 areas. Here, as a next step, we examined whether images obtained from rapid freeze-substitution (RFS) after fixation with glutaraldehyde (GA > RFS) are of comparable quality to those obtained using standard RFS. GA > RFS provided excellent preservation of mycobacterial cell ultrastructure, including visualization of cytoplasmic ribosomes, DNA fibers, and the outer membrane. The average number of ribosomes per cubic micrometer counted on RFS and GA > RFS was not significantly different (6987.8 ± 2181.0 and 6888.9 ± 1799.3, respectively). These values were higher, but not significantly so, than those obtained using conventional chemical fixation (5018.7 ± 2511.3). This procedure may be useful for RFS preparation of unculturable mycobacteria strains or virulent strains isolated in laboratories that cannot perform RFS.
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Novel freeze-substitution electron microscopy provides new aspects of virulent Mycobacterium tuberculosis with visualization of the outer membrane and satisfying Biosafety requirements.
Journal of microbiological methods, 2009Co-Authors: Hiroyuki Yamada, Satoshi Mitarai, Kinuyo Chikamatsu, Kazue Mizuno, Masashi YamaguchiAbstract:Transmission electron microscopy (TEM) of virulent bacteria is usually performed following chemical fixation (CF) with aldehyde fixatives such as glutaraldehyde because of the Biosafety problem. However, CF may alter sample ultrastructure. In this study, we used a rapid-freeze substitution (RFS) sandwich method without pre-embedding in agar. TEM images obtained using this method were completely different from those of conventional chemically fixed samples; the bacilli cytoplasm of the RFS preparations was filled evenly with numerous ribosomes, and there was no positional variation of electron density that was obvious in those obtained with CF samples. The sandwich method is suitable for microbiological materials without expensive devices and can be easily performed in a Biosafety Cabinet. In future, this method coupled with novel labeling techniques may help localize structural and functional molecules throughout a bacterial cell.
Jay Krishnan - One of the best experts on this subject based on the ideXlab platform.
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Decontamination Validation of a Class II Type A2 Biosafety Cabinet during Laboratory Fumigation
Applied Biosafety, 2020Co-Authors: Greg Frey, Cathy Robertson, Jay KrishnanAbstract:Objective:The objective of this study was to evaluate whether a Class II type A2 Biosafety Cabinet in a laboratory could be decontaminated while the laboratory was being fumigated using vaporous hy...
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Articles Decontamination of a Worst-case Scenario Class II Biosafety Cabinet Using Vaporous Hydrogen Peroxide
2012Co-Authors: Greg Fey, Stan Klassen, Steven Theriault, Jay KrishnanAbstract:The objective of this study was to evaluate both condensing (wet) and non-condensing (dry) vaporous hydrogen peroxide (VHP) technologies for decontaminating a worst-case scenario Class II Biosafety Cabinet. A 23-year-old Class II, type A2 Biosafety Cabinet equipped with loaded HEPA filters and an inoperable blower was used for this study. Biological indicators were placed at various locations within the Cabinet, including between the pleats of supply and exhaust HEPA filters, to assess the success of the decontamination processes. A variety of decontamination program cycle parameters in combination with two different routes of VHP introduction and minor Biosafety Cabinet preparations were assessed. Initial decontamination attempts using routine program cycles failed (at least one biological indicator grew upon incubation); however, program cycles consisting of modified parameters and BSC preparations resulted in successful (all the biological indicators were inactivated) and repeatable decontaminations. This study concludes that VHP, either wet or dry, could be used to decontaminate an entire Biosafety Cabinet only if appropriate and thoroughly validated decontamination processes are employed
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Decontamination of a Worst-case Scenario Class II Biosafety Cabinet using Vaporous Hydrogen Peroxide:
Applied Biosafety, 2010Co-Authors: Greg Fey, Stan Klassen, Steven Theriault, Jay KrishnanAbstract:The objective of this study was to evaluate both condensing (wet) and non-condensing (dry) vaporous hydrogen peroxide (VHP) technologies for decontaminating a worst-case scenario Class II Biosafety...
John A. Lednicky - One of the best experts on this subject based on the ideXlab platform.
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Gas-permeable ethylene bags for the small scale cultivation of highly pathogenic avian influenza H5N1 and other viruses in embryonated chicken eggs
Virology journal, 2010Co-Authors: Sara B Hamilton, Deirdre E Daniels, William Sosna, Eric R Jeppesen, Julie M Owells, Micah D. Halpern, Kimberly S. Mccurdy, Jon O. Rayner, John A. LednickyAbstract:Background Embryonated chicken eggs (ECE) are sometimes used for the primary isolation or passage of influenza viruses, other viruses, and certain bacteria. For small-scale experiments with pathogens that must be studied in Biosafety level three (BSL3) facilities, inoculated ECE are sometimes manipulated and maintained in small egg incubators within a Biosafety Cabinet (BSC). To simplify the clean up and decontamination of an egg incubator in case of egg breakage, we explored whether ethylene breather bags could be used to encase ECE inoculated with pathogens. This concept was tested by determining embryo survival and examining virus yields in bagged ECE.
Kinuyo Chikamatsu - One of the best experts on this subject based on the ideXlab platform.
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Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and freeze-substitution with osmium-acetone at ultralow temperature.
Journal of Microbiological Methods, 2013Co-Authors: Hiroyuki Yamada, Kinuyo Chikamatsu, Akio Aono, Satoshi MitaraiAbstract:Abstract Sample preparations for transmission electron microscopy of virulent Mycobacterium tuberculosis are usually performed with chemical fixation using glutaraldehyde (GA) in a Biosafety area followed by post-fixation with aqueous osmium tetroxide (OT) in a conventional laboratory outside the Biosafety area. Freeze-substitution with osmium-acetone (OA) at ultralow temperature (− 85 °C) has been shown to provide high quality final images and preserves cellular structures intact. However, some preparation procedures for freeze-substitution often require large fixed devices for freezing in a special laboratory. We have reported a novel freeze-substitution preparation method that can be performed using a portable device in a Biosafety Cabinet at Biosafety level (BSL) 3 areas. Here, as a next step, we examined whether images obtained from rapid freeze-substitution (RFS) after fixation with glutaraldehyde (GA > RFS) are of comparable quality to those obtained using standard RFS. GA > RFS provided excellent preservation of mycobacterial cell ultrastructure, including visualization of cytoplasmic ribosomes, DNA fibers, and the outer membrane. The average number of ribosomes per cubic micrometer counted on RFS and GA > RFS was not significantly different (6987.8 ± 2181.0 and 6888.9 ± 1799.3, respectively). These values were higher, but not significantly so, than those obtained using conventional chemical fixation (5018.7 ± 2511.3). This procedure may be useful for RFS preparation of unculturable mycobacteria strains or virulent strains isolated in laboratories that cannot perform RFS.
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Novel freeze-substitution electron microscopy provides new aspects of virulent Mycobacterium tuberculosis with visualization of the outer membrane and satisfying Biosafety requirements.
Journal of microbiological methods, 2009Co-Authors: Hiroyuki Yamada, Satoshi Mitarai, Kinuyo Chikamatsu, Kazue Mizuno, Masashi YamaguchiAbstract:Transmission electron microscopy (TEM) of virulent bacteria is usually performed following chemical fixation (CF) with aldehyde fixatives such as glutaraldehyde because of the Biosafety problem. However, CF may alter sample ultrastructure. In this study, we used a rapid-freeze substitution (RFS) sandwich method without pre-embedding in agar. TEM images obtained using this method were completely different from those of conventional chemically fixed samples; the bacilli cytoplasm of the RFS preparations was filled evenly with numerous ribosomes, and there was no positional variation of electron density that was obvious in those obtained with CF samples. The sandwich method is suitable for microbiological materials without expensive devices and can be easily performed in a Biosafety Cabinet. In future, this method coupled with novel labeling techniques may help localize structural and functional molecules throughout a bacterial cell.