Cryosectioning of biofilms for microscopic examination

Yu, Feipeng Philip; Callis, Gayle; Stewart, Philip S.; Griebe, Thomas; McFeters, Gordon A.

doi:10.1080/08927019409378264

Cited by 46 publications

(25 citation statements)

References 16 publications

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“…Colony biofilms were grown for 48 h on TSA lacking IPTG and then transferred to TSA supplemented with 1 mM IPTG for 4 h. An uninduced control was transferred to TSA without inducer for 4 h, and a positive control was prepared by growing the colony biofilm for 52 h in the continuous presence of IPTG. Colony biofilms were cryoembedded, and frozen sections were examined with a Nikon E800 microscope by using epifluorescent illumination (36).…”

Section: Methodsmentioning

confidence: 99%

Contributions of Antibiotic Penetration, Oxygen Limitation, and Low Metabolic Activity to Tolerance of Pseudomonas aeruginosa Biofilms to Ciprofloxacin and Tobramycin

Walters¹,

Roe²,

Bugnicourt³

et al. 2003

Antimicrob Agents Chemother

853

675

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The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either 10 g of tobramycin ml ؊1 or 1.0 g of ciprofloxacin ml ؊1 . After 100 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 ؎ 0.18 for tobramycin and 1.42 ؎ 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 m into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.

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Section: Methodsmentioning

confidence: 99%

Contributions of Antibiotic Penetration, Oxygen Limitation, and Low Metabolic Activity to Tolerance of Pseudomonas aeruginosa Biofilms to Ciprofloxacin and Tobramycin

Walters¹,

Roe²,

Bugnicourt³

et al. 2003

Antimicrob Agents Chemother

853

675

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show abstract

“…compound (Miles, Inc., Elkhart, Ind.) for 15 min and then freezing them on dry ice (18,60). Once completely frozen, the biofilm was removed from the ceramic coupon, turned over, placed back on dry ice, and reembedded in O.C.T.…”

Section: Methodsmentioning

confidence: 99%

Effects of Current Velocity on the Nascent Architecture of Stream Microbial Biofilms

Battin

Kaplan

Newbold

et al. 2003

Appl Environ Microbiol

258

200

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Current velocity affected the architecture and dynamics of natural, multiphyla, and cross-trophic level biofilms from a forested piedmont stream. We monitored the development and activity of biofilms in streamside flumes operated under two flow regimes (slow [0.065 m s ؊1 ] and fast [0.23 m s ؊1 ]) by combined confocal laser scanning microscopy with cryosectioning to observe biofilm structure and composition. Biofilm growth started as bacterial microcolonies embedded in extracellular polymeric substances and transformed into ripple-like structures and ultimately conspicuous quasihexagonal networks. These structures were particularly pronounced in biofilms grown under slow current velocities and were characterized by the prominence of pennate diatoms oriented along their long axes to form the hexagons. Microstructural heterogeneity was dynamic, and biofilms that developed under slower velocities were thicker and had larger surface sinuosity and higher areal densities than their counterparts exposed to higher velocities. Surface sinuosity and biofilm fragmentation increased with thickness, and these changes likely reduced resistance to the mass transfer of solutes from the water column into the biofilms. Nevertheless, estimates of dissolved organic carbon uptake and microbial growth suggested that internal cycling of carbon was more important in thick biofilms grown in slow flow conditions. High-pressure liquid chromatography-pulsed amperometric detection analyses of exopolysaccharides documented a temporal shift in monosaccharide composition as the glucose levels decreased and the levels of rhamnose, galactose, mannose, xylose, and arabinose increased. We attribute this change in chemical composition to the accumulation of diatoms and increased incorporation of detrital particles in mature biofilms.

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“…; moreover its tetrazolium salts, as 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride and 5-cyano-2,3-ditolyl tetrazolium chloride, are very important in view of their use as indicators of respiratory activity and cytotoxicity assays. 2,3 Theoretically the following two tautomers (A and B) can be expected for formazan :…”

Section: Introductionmentioning

confidence: 99%

Ab initio study of formazan and 3-nitroformazan

Buemi¹,

Zuccarello²,

Venuvanalingam³

et al. 1998

Faraday Trans.

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Formazan and 3-nitroformazan have been investigated at ab initio level (MP2/6-31G** and B3LYP/6-31G**) in all their possible conformations, for studying the various possibilities of intramolecular hydrogen bonding formation. The trans-syn-s-cis (TSSC), known also as yellow form, has been found to be the most stable conformer (at least in the gas phase) in both compounds. This particular structure is strongly stabilized by a NwHÉ É ÉN hydrogen bridge, which gives rise to a hexatomic chelate ring, with the possibility of a proton transfer process.This closely resembles that of malondialdehyde, previously studied, in the evolution of the potential energy shape but with a greater barrier height. Various approaches for obtaining a quantitative estimate of the energy of the hydrogen bridges are discussed. The electronic structures of the most favoured TSSC, TSST (trans-syn-s-trans) and TAST (trans-anti-s-trans) conformations of formazan have been compared with those of the corresponding forms of 1,5-diphenylformazan, in order to account for the UV spectra available in the literature and the di †erent colours exhibited by the molecule on passing from one conformation to another.

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Cryosectioning of biofilms for microscopic examination

Cited by 46 publications

References 16 publications

Contributions of Antibiotic Penetration, Oxygen Limitation, and Low Metabolic Activity to Tolerance of Pseudomonas aeruginosa Biofilms to Ciprofloxacin and Tobramycin

Contributions of Antibiotic Penetration, Oxygen Limitation, and Low Metabolic Activity to Tolerance of Pseudomonas aeruginosa Biofilms to Ciprofloxacin and Tobramycin

Effects of Current Velocity on the Nascent Architecture of Stream Microbial Biofilms

Ab initio study of formazan and 3-nitroformazan

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