Biofilm thickness measurements by light microscopy

Light microscopy has been used to measure biofilm thickness. The vertical displacement of the sample required to focus from the biofilm-liquid interface to the biofilm-substratum interface is measured by the stage micrometer. Biofilm thickness is proportional, but not equal, to the measured vertical displacement. An expression for the proportionality constant, k_f, in terms of refractive indices is determined from a geometric analysis of the light path. k_f can be estimated as the ratio of the refractive index of the film to the refractive index of the media interfacing the film between the objective lens and the sample. The thickness of any transparent film may be determined by light microscopy when the refractive index of the film is known.     

Quantitative analysis of biofilm thickness variability

The thickness variability of biofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the binary population combination of these two species was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and applying image analysis to construct thickness profiles along linear transects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell attached to the surface (<5 mum) and as thick as 1000 mum. Week-old biofilms of three different species compositions displayed distinct structural features as indicated by their mean thicknesses and by a roughness coefficient. Monopopulation biofilms of P. aeruginosa (29 mum mean thickness) or K. pneumoniae (100 mum mean thickness) were thinner than the binary population biofilm (400 mum mean thickness). A roughness coefficient developed in this investigation corroborated the qualitative visual characterization of P. aeruginosa biofilms as relatively uniformly thick (mean roughness coefficient 0.15), K. pneumoniae biofilms as patchy (mean roughness coefficient 1.14), and the binary population biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum completely, significant areas of essentially bare substratum were apparent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilms may be due to the fact that this organism is nonmotile. A spatial correlation analysis of the thickness data indicated that thickness measurements were still correlated even when separated by distances that exceeded the mean biofilm thickness. Cell aggregates, some of them hundreds of microns in size, were observed in the effluent of K. pneumoniae and binary population biofilm reactors. Measurements of thickness variability and other observations reported in this article provide a quantitative basis for analysis of microscale structural heterogeneity of biofilms. (c) 1995 John Wiley & Sons, Inc.     

Measurement of spinal sagittal curvatures using the laser triangulation method

The purpose of the first part of the study was to establish the variability of repeated measurements in different measuring conditions. In the second part, we performed in a large number of patients, a measurement of thoracic kyphosis and lumbar lordosis and compared them to age, gender, and level of nourishment. In the first part, measurements were performed on a plastic model of the back of a patient with a rigid and a normal spine. In the second part, 250 patients participated in the study (126 men and 124 women). For measuring spinal curvatures we used an apparatus for laser triangulation constructed at the Faculty of Mechanical Engineering, University of Ljubljana. A comparison of 30 repeated measurements was shown as the average value +/- 2 SD which included 95% of the results. Thirty repeated readings of one 3D measurement: thoracic kyphosis 41.2 degrees +/- 0.6 degrees, lumbar lordosis 4.4 degrees +/- 1.2 degrees; 30 measurements on a plastic model: thoracic kyphosis 36.8 degrees +/- 1.2 degrees, lumbar lordosis 30.9 degrees +/- 2.0 degrees; 30 measurements on a patient with a rigid spine: thoracic kyphosis 41.5 degrees +/- 2.4 degrees, lumbar lordosis 4.0 degrees +/- 1.8 degrees; 30 measurements on a patient with a normal spine: thoracic kyphosis 48.8 degrees +/- 7.4 degrees, lumbar lordosis 21.1 degrees +/- 4.4 degrees. The average size of thoracic kyphosis in 250 patients was 46.8 degrees (SD 10.1 degrees) and lumbar lordosis 31.7 degrees (SD 12.5 degrees). The angle size was statistically significantly correlated to gender (increased thoracic kyphosis and lumbar lordosis in women) and body mass index (increased thoracic kyphosis and lumbar lordosis in more nourished patients). Age was not significantly correlated to the observed angles. During measurements of the spinal angles it was important to pay attention to relaxation and the patient's position as well as to perform more measurements providing the average value. The age and the level of nourishment influence the size of the sagittal spinal angles. In the observed sample the effect of age was not confirmed.     

Biofilm diatom community structure: Influence of temporal and substratum variability

Abstract

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

Biofilm diatom community structure: influence of temporal and substratum variability

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

Interferometric analysis of intrasection and intersection thickness variability associated with cryostat microtomy

Summary Mach-Zehnder interferometric measurements were used to assess the extent of section thickness variability (inter- and intrasection) associated with cryostat microtomy of adrenal sections over a typical working range of 10–20 m. Sections were obtained using a Bright's Cambridge rocking type and a Damon rotary type cryostat microtome to allow comparative analyses. The effective thickness of tissue sections after being mounted onto slides by flash drying was reduced by 90% relative to microtome section thickness setting. A linear relationship between measured thickness and microtome setting was obtained with both instruments. Thickness variability between replicate sections over the range of microtome settings approximated 11% for the rocking microtome and 5% with the rotary microtome. Average intrasection variability was found to be 7% for rocking microtome sections and 4% for sections obtained with the rotary microtome. However, this variability is a negligible source of error in cytophotometric analyses, providing replicate sections are used and an adequate number of measurements are made on mask-delimited individual cells or tissue specimen areas.     

On-line monitoring of hybridoma cell growth using a laser turbidity sensor

A high-sensitivity turbidity probe was used for on-line monitoring of the cell concentration in batch hybridoma cultivation. Good correlation between off-line cell counts and the linearized sensor signal was found. The quality of the signal was sufficiently high to provide for on-line estimation of the specific growth rate using an efficient filtering procedure. These positive results suggest that such laser turbidity sensors will facilitate development of systems for on-line monitoring and control of animal cell cultivations. (c) 1992 John Wiley & Sons, Inc.     

Destruction of a Bacterial Biofilm with an Electrochemically Activated Solution

Biophysics - The action of electrochemically activated water on the fine structure of biofilms formed by the plankton forms of lactic acid bacteria and E. coli was investigated. Bacterial biofilms...     

Biofilm thickness effect on the diffusion limitation in the bioprocess reaction: Biofloc critical diameter significance

A convenient physical model for biomass involves consideration of individual cells as active centers dispersed through a continuous region in which transport takes place by molecular diffusion. This paper investigates for steady state conditions the variation of apparent kinetic constant (K _m ) of bacteria in relation to biofloc diameter with solid and/or liquid-phase diffusion. When the biochemical reactions are limited only by liquid-phase diffusion, theK _m increases whenD increases. With solid-phase diffusion limitation only, theK _m increases linearly with the diameterD of the floc. When both solid and liquid-phase diffusion limitations are considered, the apparentK _m is affected by liquid-phase diffusion limitation with very smallD and by solid-phase diffusion limitation with higherD. The critical diameterD _c can be assumed to be theD at which solid-phase diffusion limitation becomes more significant than liquid-phase diffusion limitation.     

Planktonic-Cell Yield of a Pseudomonad Biofilm

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10⁹ cells ml⁻¹, suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.     

Comparison of variability in wall thickness for sex determination of cremated cadavers

The amount of information provided by cremations is primarily determined by the general state of preservation. Consequently the possibilities of sex determination by means of morphognostic methods are limited due to a mostly poor representation of fragments which can be of diagnostic value. Therefore an improval of methods has to focus on those skeletal elements that occur in every or almost every batch. These are in particular fragments of the diaphyses and the cranial vault. Wall thicknesses are measured and the values of those individuals that cannot be sexed by conventional methods are compared to an intragroup reference. Following the principle of the morphognostic series about 25% of previously not sexable individuals can now be assigned.     

Phenotypic variability and correlation between skinfold thickness and arterial pressure level]

Contribution of genetic and environmental factors in phenotypic variability of blood pressure level and skinfold thickness, and phenotypic correlation between these characters was calculated on the basis of familial correlations. It was shown that genetic determinant explains considerable portion of blood pressure level and skinfold thickness variability. Among common environmental effects, the factors affecting one generation are important with regard to variability of these characters. Maternal effect is expressed in the variability of systolic and diastolic blood pressure. Correlation between blood pressure level and triceps skinfold thickness is determined by genetic factors, whereas that between blood pressure level and subscapular skinfold thickness is mediated by environmental factors. The results obtained may be applied in populational prevention of cardiovascular disease.     

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO₂ sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.     

Hydrogen/deuterium exchange and aggregation of a polyvaline and a polyleucine alpha-helix investigated by matrix-assisted laser desorption ionization mass spectrometry

The membrane-associated pulmonary surfactant protein C (SP-C), containing a polyvaline alpha-helix, and a synthetic SP-C analogue with a polyleucine helix (SP-C(Leu)) were studied by hydrogen/deuterium exchange matrix-assisted laser desorption ionization (MALDI) mass spectrometry. SP-C, but not SP-C(Leu), formed abundant amyloid fibrils under experimental conditions. In CD(3)OD/D(2)O, 91:9 (v/v), containing 2 mM ammonium acetate, SP-C(Leu) and SP-C exchanged 40% of their exchangeable hydrogens within 1 min. This corresponds to exchange of labile side-chain hydrogen atoms, hydrogens on the N- and C-terminal heteroatoms, and amide hydrogen atoms in the unstructured N-terminal regions. After approximately 300 h, four exchangeable hydrogen atoms in SP-C(Leu) and 10 in SP-C remained unexchanged. During this time period the ion current corresponding to singly charged SP-C decreased to <10% of the initial value due to the formation of insoluble aggregates that are not detected by MALDI mass spectrometry. In contrast, the ion current for SP-C(Leu) was maintained over this time period, although the peptides were incubated together. In combination, hydrogen/deuterium exchange and aggregation data indicate that the polyleucine peptide refolds into a helix after opening, while the unfolded polyvaline peptide forms insoluble beta-sheet aggregates rather than refolding into a helix. The SP-C helix, but not the SP-C(Leu) helix, is thus in a metastable state, which may contribute to the recently observed tendency of SP-C and its precursor to misfold and aggregate in vivo.     

Poliovirus-1 Inactivation and Interaction with Biofilm: a Pilot-Scale Study

A pilot-scale study was initiated to examine the behavior of viruses pulse injected into a distribution system. The influence of a free-chlorine residual and that of virus preadsorption to clay particles was evaluated by tracing the viruses both in the water flow and after elution from the biofilm. These experiments demonstrated, first, that virus preadsorption on 40 mg of Na-montmorillonite per liter increased the residence time of the viruses within the pilot plant by roughly three times and, second, that preadsorption to clay did not prevent viruses from being inactivated by chlorine. Moreover, with no clay added, a greater amount of viruses was recovered from the biofilm than from the water flow (by a factor of 2 or 10 in the absence or presence of chlorine, respectively), indicating a tendency for virus accumulation within biofilms.     

Presence of a Polymicrobial Endometrial Biofilm in Patients with Bacterial Vaginosis

Objective

To assess whether the bacterial vaginosis biofilm extends into the upper female genital tract.

Study Design

Endometrial samples obtained during curettage and fallopian tube samples obtained during salpingectomy were collected. Endometrial and fallopian tube samples were analyzed for the presence of bacteria with fluorescence-in-situ-hybridisation (FISH) analysis with probes targeting bacterial vaginosis-associated and other bacteria.

Results

A structured polymicrobial Gardnerella vaginalis biofilm could be detected in part of the endometrial and fallopian tube specimens. Women with bacterial vaginosis had a 50.0% (95% CI 24.0–76.0) risk of presenting with an endometrial Gardnerella vaginalis biofilm. Pregnancy (AOR  = 41.5, 95% CI 5.0–341.9, p<0.001) and the presence of bacterial vaginosis (AOR  = 23.2, 95% CI 2.6–205.9, p<0.001) were highly predictive of the presence of uterine or fallopian bacterial colonisation when compared to non-pregnant women without bacterial vaginosis.

Conclusion

Bacterial vaginosis is frequently associated with the presence of a structured polymicrobial Gardnerella vaginalis biofilm attached to the endometrium. This may have major implications for our understanding of the pathogenesis of adverse pregnancy outcome in association with bacterial vaginosis.