Effects of SEM Preparation Techniques on the Appearance of Bacteria and Biofilms in the Carter Sandstone

Fratesi, Sarah E.; Lynch, F. Leo; Kirkland, Brenda L.; Brown, Lewis R.

doi:10.1306/042604740858

Cited by 103 publications

(63 citation statements)

References 35 publications

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“…After 24 h, slides were washed with 1ϫ PBS (three times) to remove planktonic cells. The slides were prepared for scanning electron microscopy (SEM) using a previously published protocol (17). The slides were placed in 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) for 2 h. The slides were then dehydrated by successive 10-min incubations in 35% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol followed by six successive 10-min incubations in 100% ethanol.…”

Section: Methodsmentioning

confidence: 99%

Candida albicans and Staphylococcus aureus Form Polymicrobial Biofilms: Effects on Antimicrobial Resistance

Harriott

Noverr

2009

Antimicrob Agents Chemother

488

399

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Candida albicans readily forms biofilms on the surface on indwelling medical devices, and these biofilms serve as a source of local and systemic infections. It is estimated that 27% of nosocomial C. albicans bloodstream infections are polymicrobial, with Staphylococcus aureus as the third most common organism isolated in conjunction with C. albicans. We tested whether S. aureus and C. albicans are able to form a polymicrobial biofilm. Although S. aureus formed poor monoculture biofilms in serum, it formed a substantial polymicrobial biofilm in the presence of C. albicans. In terms of architecture, S. aureus formed microcolonies on the surface of the biofilm, with C. albicans serving as the underlying scaffolding. In addition, S. aureus matrix staining revealed a different phenotype in polymicrobial versus monomicrobial biofilms, suggesting that S. aureus may become coated in the matrix secreted by C. albicans. S. aureus resistance to vancomycin was enhanced within the polymicrobial biofilm, required viable C. albicans, and was in part mediated by C. albicans matrix. However, the growth or sensitivity to amphotericin B of C. albicans is not altered in the polymicrobial biofilm.

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Section: Methodsmentioning

confidence: 99%

Candida albicans and Staphylococcus aureus Form Polymicrobial Biofilms: Effects on Antimicrobial Resistance

Harriott

Noverr

2009

Antimicrob Agents Chemother

488

399

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“…, with HMDS, or critical point drying) also resulted in significant, visible differences in the appearance of the dehydrated biofilm, particularly at the nanometer scale, including tiny, spherical artifacts [5]. Dehydration in acetone produced the best-preserved biofilm structure for our TEM sections.…”

mentioning

confidence: 88%

“…Therefore, we strive to image only freshly broken surfaces and we use acid only to prepare samples when we can compare an acid-etched surface to a freshly broken surface. Trials of different dehydration methods (air drying, air drying under vacuum, sequential ethyl alcohol solutions, with HMDS, or critical point drying) also resulted in significant, visible differences in the appearance of the dehydrated biofilm, particularly at the nanometer scale, including tiny, spherical artifacts [5]. Dehydration in acetone produced the best-preserved biofilm structure for our TEM sections.…”

mentioning

confidence: 90%

Nannobacteria, Organic Matter, and Precipitation in Hot Springs, Viterbo, Italy: Distinctions and Relevance

Kirkland¹,

Lynch

Folk

et al. 2008

Microscopy Today

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Tiny (50-200 nm) spheroids were first discovered by Folk through SEM work on the hot springs of Viterbo Italy. He termed these small, spherical structures "nannobacteria, " and proposed that they may be important agents in precipitation of CaCO 3 , as needle-like crystals of the mineral aragonite, and as bundles of such needle-like crystals (termed "fuzzy dumbbells"), or as elongated crystals of the mineral calcite [1].During the past 15 years, nanometer-scale spheroids have been discovered in the geological, medical, and astronomical worlds. There can be no doubt as to their existence, but their significance and origin remain a subject of continuing controversy. Even the spelling ("nanno-"), which has been the standard in biology, geology, and paleontology going back to the 19 th century, has been questioned. Whether or not they are truly bacteria or any form of life has been a subject of heated debate. At one end of the spectrum are claims that they are a minute form of microbial life (bacteria or archaea) because they are culturable organisms with distinct cell walls/membranes that stain (+) for DNA. Other workers suggest that they are membrane vesicles blebbing off of the cell walls of "normal" bacteria. Another view is that they represent inchoate mineral nuclei formed either in the presence of bacterial slime (EPS) or in other organic-rich milieus. A key argument of opponents to the concept of very small bacteria is that a minimum volume of 200 nm 3 is thought to be needed to hold all the molecules essential for life functions [2]. These debates have motivated years of research. If nothing else, we have learned that when trying to answer questions at the nanometer scale, careful sample preparation is essential. Unfortunately, during preparation of samples for SEM, spherical nannobacteria-like artifacts can be produced by gold coating for more than 60 seconds. Therefore, we coat our samples for no more than 30 seconds to avoid creation of spherical artifacts [3]. Unfortunately, sub-spherical, nannobacteria-like features can also be produced by acid-etching of purely inorganic minerals [4]. Therefore, we strive to imageonly freshly broken surfaces and we use acid only to prepare samples when we can compare an acid-etched surface to a freshly broken surface. Trials of different dehydration methods (air drying, air drying under vacuum, sequential ethyl alcohol solutions, with HMDS, or critical point drying) also resulted in significant, visible differences in the appearance of the dehydrated biofilm, particularly at the nanometer scale, including tiny, spherical artifacts [5]. Dehydration in acetone produced the best-preserved biofilm structure for our TEM sections. Finally, experiments in which bacterial cultures were lysed and then air-dried resulted in formation of bacterial debris containing nanometer-scale textures [4].With the remains of lysed bacteria in mind, we established criteria for distinguishing nannobacteria from nanometer-scale textures in fine-scale organic debris. In SEM, nannobacteria appear...

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“…The fact that AFM is compatible with the aqueous environment means that artifacts caused by drying and/or coating of samples for scanning electron microscopy (SEM) can be avoided (Drake et al 1989;Fratesi et al 2004;Müller & Dufrêne 2008), and it permits the experimenter to maintain biological materials in physiologically relevant conditions (Pelling et al 2004;Cross et al 2007). Moreover, unlike SEM images, AFM topographs provide precise information about the three-dimensional organization of cellular ultrastructure (Allison & Doktycz 2006;Radmacher 2007).…”

Section: Introductionmentioning

confidence: 99%

Fractal analysis of cell boundary ultrastructure imaged by atomic force microscopy

Kim

et al. 2015

Animal Cells and Systems

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Fractal analysis has been widely used to determine the complexity of irregular shapes of various natural objects including biological systems. Here we report an investigation into the nature of the cell boundary nanostructure (less than 200 nm in thickness) in an aqueous environment using atomic force microscopy. The box-counting method revealed the fractal nature of detailed contours of cells cultured on a solid substratum. The fractal dimensions (D f ) of individual cells of the human epithelial cell line MCF10A converged on a narrow range (D f = 1.352 ± 0.044) despite their morphological diversity. Treatment with cytochalasin D, an inhibitor of actin polymerization, was shown to decrease both the ruggedness and the D f of cell boundaries. These findings suggest that the D f value is a useful parameter for describing the complexity of cellular ultrastructure dependent on the actin cytoskeleton.

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Effects of SEM Preparation Techniques on the Appearance of Bacteria and Biofilms in the Carter Sandstone

Cited by 103 publications

References 35 publications

Candida albicans and Staphylococcus aureus Form Polymicrobial Biofilms: Effects on Antimicrobial Resistance

Candida albicans and Staphylococcus aureus Form Polymicrobial Biofilms: Effects on Antimicrobial Resistance

Nannobacteria, Organic Matter, and Precipitation in Hot Springs, Viterbo, Italy: Distinctions and Relevance

Fractal analysis of cell boundary ultrastructure imaged by atomic force microscopy

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