Image contrast of whole bacteria was compared in Staphylococcus aureus and Escherichia coli depending on pre-stain suspension liquids by energy-filtering transmission electron microscopy. The two bacterial strains were suspended in three most commonly used liquids for negative staining (triple disti...
Image contrast of whole bacteria was compared in Staphylococcus aureus and Escherichia coli depending on pre-stain suspension liquids by energy-filtering transmission electron microscopy. The two bacterial strains were suspended in three most commonly used liquids for negative staining (triple distilled water [DW], phosphate-buffered saline [PBS], and nutrient broth [NB]) and directly observed without staining or stained with neutralized potassium phosphotungstate (PTA), respectively. Even though in low contrast, unstained bacteria were observed owing to their inherent electron density and cell shape in zero-loss (elastic scattering) images. After being suspended in PBS, unstained bacteria appeared to have higher contrast and more refined periphery than DW-suspended ones, and extracellular appendage structures such as fimbriae and flagella could be discerned. The unstained bacteria appeared to be invariably surrounded with electron-lucent precipitates, possibly from PBS. As far as delineation of the structures, the combination of DW or PBS suspension with subsequent staining provided the most satisfactory results, as evidenced by the high contrast of bacterial morphology and appendage structures. However, after being suspended in NB and stained with PTA, bacteria often had too high contrast or poor staining, with electron-dense aggregates around the bacteria. These results suggest that suspension with concentrated organic aliquots including broth media before PTA staining could deteriorate image contrast, and should be used only in dilute form for visualizing bacterial morphology and appendage structures. Moreover the contrast enhancement of unstained bacteria by salt granules would be advantageous in demonstrating bacterial sorption of environmental particles like heavy metals, maintaining minimal contrast for cell imaging.
Image contrast of whole bacteria was compared in Staphylococcus aureus and Escherichia coli depending on pre-stain suspension liquids by energy-filtering transmission electron microscopy. The two bacterial strains were suspended in three most commonly used liquids for negative staining (triple distilled water [DW], phosphate-buffered saline [PBS], and nutrient broth [NB]) and directly observed without staining or stained with neutralized potassium phosphotungstate (PTA), respectively. Even though in low contrast, unstained bacteria were observed owing to their inherent electron density and cell shape in zero-loss (elastic scattering) images. After being suspended in PBS, unstained bacteria appeared to have higher contrast and more refined periphery than DW-suspended ones, and extracellular appendage structures such as fimbriae and flagella could be discerned. The unstained bacteria appeared to be invariably surrounded with electron-lucent precipitates, possibly from PBS. As far as delineation of the structures, the combination of DW or PBS suspension with subsequent staining provided the most satisfactory results, as evidenced by the high contrast of bacterial morphology and appendage structures. However, after being suspended in NB and stained with PTA, bacteria often had too high contrast or poor staining, with electron-dense aggregates around the bacteria. These results suggest that suspension with concentrated organic aliquots including broth media before PTA staining could deteriorate image contrast, and should be used only in dilute form for visualizing bacterial morphology and appendage structures. Moreover the contrast enhancement of unstained bacteria by salt granules would be advantageous in demonstrating bacterial sorption of environmental particles like heavy metals, maintaining minimal contrast for cell imaging.
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제안 방법
The contrast tuning or enhancement by decoration of bacteria using PBS or similar buffered salines would be advantageous in demonstrating bacterial interactions with fine-grained heavy metals, while maintaining minimal contrast for cell imaging. After being stained with conventional negative stains, the PBS-suspended bacterial surfece would be covered with metal stains and extraneously treated cell-bound heavy metals, making them difficult to be discriminated based on their electron density, as inferred in this study (Figs. 2C and 2D; 4C and 4D). The discrimination may need significant levels of defocus and consequent contrast transfer function correction.
It was affirmed in this study that the combination ofDW or PBS suspension with subsequent PTA staining provided the most satisfactory results. It remains to be validated the effects of pre-stain suspension liquids on diverse bacterial strains and other negative stains such as uranyl acetate and ammonium molybdate in order to confirm their broad-spectrum characteristics and establish optimal staining regimes in further studies. The presently proposed "low contrast” negative stain for bacteria, a simple and rapid decoration procedure for demonstrating bacteria using PBS precipitates, would facilitate characterization of diverse clinical or environmental isolates that are reactive with metal ions present in the external milieu.
This study provided technical precautions to be considered before conventional negative staining of cultured bacteria. There were significant differences in image contrast depending on suspension liquids before PTA staining.
대상 데이터
These results suggest that suspension with concentrated organic aliquots including broth media before PTA staining could deteriorate image contrast, and should be used only in dilute form for visualizing bacterial morphology and appendage structures. The three types of pre-stain suspension liquids assayed in this study are the three most commonly used liquids that are widely available m microbiology laboiatories and are easily selected by workers. Therefore, the results in this study might serve as a practical reference in selecting pre-suspension liquids when using PTA for conventional negative staining of bacteria.
후속연구
) could generate higher amounts of inelastically scattered electrons than the DW-or PBS-suspended treatments, which cause the decrease of elastically scattered electrons and subsequent contrast deterioration in images. However, this study does not exclude the possibility that components of NB act like a surfactant that results in partial or poor staining of bacterial cells and extracellular appendage structures by PTA, In addition, the organic components of NB may react undesirably with PTA to fonn electron-dense aggregates around the bacteria and interfere with the negative staining procedure, leading to the consequent partial or poor staining of the bacteria. The different stainhi용 profiles between the two bacterial strains remain to be investigated in further studies.
The three types of pre-stain suspension liquids assayed in this study are the three most commonly used liquids that are widely available m microbiology laboiatories and are easily selected by workers. Therefore, the results in this study might serve as a practical reference in selecting pre-suspension liquids when using PTA for conventional negative staining of bacteria.
참고문헌 (24)
Chavez, F. P., H. Lunsdorf, and C. A. Jerez. 2004. Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate. Appl. Environ. Microbiol. 70: 3064-3072
Fitzhenry, R., S. Dahan, A. G. Torres, Y. Chong, R. Heuschkel, S. H. Murch, et al. 2006. Long polar fimbriae and tissue tropism in Escherichia coli O157:H7. Microb. Infect. 8: 1741-1749
Glasauer, S., S. Langley, and T. J. Beveridge. 2001. Sorption of Fe (hydr)oxides to the surface of Shewanella putrefaciens: Cellbound fine-grained minerals are not always formed de novo. Appl. Environ. Microbiol. 67: 5544-5550
Hamilton, R. C., J. Bennet, D. Drane, E. Pietrzykowski, F. Seddon, A. Stefancic, and J. Cox. 1994. Negative staining can cause clumping of Bordetella pertussis fimbriae. Micron 25: 613-615
Harris, J. R. and R. W. Horne. 1994. Negative staining: A brief assessment of current technical benefits, limitations and future possibilities. Micron 25: 5-13
Hayat, M. A. 2000. Principles and Techniques of Electron Microscopy: Biological Applications, pp. 367-399. 4th Ed. Cambridge University Press, Cambridge
Jung, W. K., H. C. Koo, K. W. Kim, S. Shin, S. H. Kim, H. Yang, and Y. H. Park. 2008. Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli. Appl. Environ. Microbiol. 74: 2171-2178
Kaneko, Y., R. Danev, K. Nagayama, and H. Nakamoto. 2006. Intact carboxysomes in a cyanobacterial cell visualized by Hilbert differential contrast transmission electron microscopy. J. Bacteriol. 188: 805-808
Kohler, T., L. K. Curty, F. Barja, C. Van Delden, and J.-C. Pechere. 2000. Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J. Bacteriol. 182: 5990-5996
Kim, J. S., J. H. Chang, S. I. Chung, and J. S. Yum. 1999. Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility. J. Bacteriol. 181: 6969-6976
Ludi, S., J. Frey, D. Favre, and M. H. Stoffel. 2006. Assessing the expression of enterotoxigenic Escherichia coli-specific antigens in recombinant strains by transmission electron microscopy and immunolabeling. J. Histochem. Cytochem. 54: 473-477
Lunsdorf, H., I. Kristen, and E. Barth. 2006. Cationic hydrous thorium dioxide colloids-a useful tool for staining negatively charged surface matrices of bacteria for use in energy-filtered transmission electron microscopy. BMC Microbiol. 6: 59-66
Lutz-Meindl, U. 2007. Use of energy filtering transmission electron microscopy for image generation and element analysis in plant organisms. Micron 38: 181-196
Massover, W. H. and P. Marsh. 2000. Light atom derivatives of structure-preserving sugars are unconventional negative stains. Ultramicroscopy 85: 107-121
Mavrocordatos, D., W. Pronk, and M. Boller. 2004. Analysis of environmental particles by atomic force microscopy, scanning and transmission electron microscopy. Water Sci. Technol. 50: 9-18
Pal, S., Y. K. Tak, and J. M. Song. 2007. Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the Gram-negative bacterium Escherichia coli. Appl. Environ. Microbiol. 73: 1712-1720
Remonsellez, F., A. Orell, and C. A. Jerez. 2006. Copper tolerance of the thermoacidophilic archaeon Sulfolobus metallicus: Possible role of polyphosphate metabolism. Microbiology 152: 59-66
Saad, M. M., H. Kobayashi, C. Marie, I. R. Brown, J. W. Mansfield, W. J. Broughton, and W. J. Deakin. 2005. NopB, a type III secreted protein of Rhizobium sp. strain NGR 234, is associated with pilus-like surface appendages. J. Bacteriol. 187: 1173-1181
Scheid, P., L. Kempster, U. Griesenbach, J. C. Davies, A. Dewar, P. P. Weber, et al. 2001. Inflammation in cystic fibrosis airways: Relationship to increased bacterial adherence. Eur. Respir. J. 17: 27-35
Shieh, W. Y., A. L. Chen, and H. H. Chiu. 2000. Vibrio aerogenes sp. nov., a facultatively anaerobic marine bacterium that ferments glucose with gas production. Int. J. Sys. Evol. Microbiol. 50: 321-329
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