Spatial distributions of copper in microbial biofilms by scanning electrochemical microscopy

The spatial distribution of Cu was determined in Escherichia coli PHL628 biofilms using a scanning electrochemical microscope (SECM) consisting of a microelectrode in conjunction with a piezoelectric micropositioning system. Aqueous labile copper species were determined using voltametric stripping after reductive deposition of Cu for 4 min on the microelectrode at -0.7 V (vs Ag/AgCl). The position of the bulk solution-biofilm interface was determined from the change in current produced by 0.4 mM hydroxymethyl ferrocene that was added as a redox indicator. After a 2 h exposure to 0.2 mM copper, Cu was located in the upper region of the biofilm with a penetration depth less than 150 microm. A one-dimensional diffusive transport model adequately described the spatial distribution of copper in the biofilm, but the Cu retardation factor in the biofilm was more than 6-fold larger than that calculated from the isotherm for Cu binding to suspensions of E. coli PHL628 cells. There are several possible reasons for this difference, including an increase in the amount of extracellular polymer per cell within the biofilm and/or tortuosity that might hinder Cu transport into biofilms. The SECM technique in combination with model calculations provides direct evidence in support of the concept that formation of a biofilm may confer resistance to transient spikes in the bulk solution concentration of toxic metal species by retarding metal diffusion and reducing the metal exposure of cells within the biofilm.     

Investigation of fish product–metal container interaction using scanning electron microscopy–X-ray microanalysis

Scanning electron microscopy (SEM) and X-ray microanalysis (EDS) were used to investigate metal can discoloration and lacquer adhesion failure in enameled food cans containing tuna in vegetable oil and octopus in brine, respectively. Black and brown spots on the internal surface of the can body were caused by the formation of FeS and SnS, respectively. The source of metal can discoloration was traced to inadequate lacquering of the tin plated steel, exposing both tin and iron to sulfur containing amino acids originating from the tuna product. Enamel adhesion failure in canned octopus was also traced to local defects in the lacquer coating enabling both NaCl and citric acid contained in the brine to cause enamel blisters on the can body leading to lacquer peeling and in turn to local detinning and steel corrosion. The first defect known as “sulfide staining” is harmless to human health and does not usually affect the product. The second defect may cause extensive detinning and steel corrosion possibly affecting the safety of the canned product. This postulation is supported by iron and tin concentrations both in the product and liquid medium carried out by atomic absorption spectroscopy.     

Application of high-angle annular dark field scanning transmission electron microscopy,scanning transmission electron microscopy-energy dispersive X-ray spectrometry,and energy-filtered transmission electron microscopy to the characterization of nanoparticles in the environment

A major challenge to the development of a fundamental understanding of transport and retardation mechanisms of trace metal contaminants (<10 ppm) is their identification and characterization at the nanoscale. Atomic-scale techniques, such as conventional transmission electron microscopy, although powerful, are limited by the extremely small amounts of material that are examined. However, recent advances in electron microscopy provide a number of new analytical techniques that expand its application in environmental studies, particularly those concerning heavy metals on airborne particulates or water-borne colloids. High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM), STEM-energy-dispersive X-ray spectrometry (EDX), and energy-filtered TEM (EFTEM) can be effectively used to identify and characterize nanoparticles. The image contrast in HAADF-STEM is strongly correlated to the atomic mass: heavier elements contribute to brighter contrast. Gold nanocrystals in pyrite and uranium nanocrystals in atmospheric aerosols have been identified by HAADF-STEM and STEM-EDX mapping and subsequently characterized by high-resolution TEM (HRTEM). EFTEM was used to identify U and Fe nanocrystals embedded in an aluminosilicate. A rare, As-bearing nanophase, westerveldite (FeAs), was identified by STEM-EDX and HRTEM. The combined use of these techniques greatly expands the effective application of electron microscopy in environmental studies, especially when applied to metals of very low concentrations. This paper describes examples of how these electron microbeam techniques can be used in combination to characterize a low concentration of heavy metals (a few ppm) on nanoscale particles.     

Chemical heterogeneity of organic soil colloids investigated by scanning transmission X-ray microscopy and C-1s NEXAFS microspectroscopy

Colloid release and deposition in soils and sorption of inorganic and organic pollutants to soil colloids are strongly influenced by the composition and chemical heterogeneity of colloidal soil particles. To investigate the chemical heterogeneity of organic soil colloids at the particle scale, we used synchrotron scanning transmission X-ray microscopy (STXM) and C-1s near-edge X-ray absorption fine structure (NEXAFS) spectroscopy on 49 individual particles isolated from the surface horizons of three forest soils. Stacks of 130 images of each particle were collected at different X-ray energies between 280 and 310 eV. From these image arrays, NEXAFS spectra were obtained for each pixel and analyzed by principle component analysis and cluster analysis (PCA-CA) to characterize the intraparticle heterogeneity of the organic components. The results demonstrate that the organic matter associated with water-dispersible soil colloids is chemically heterogeneous at the single-particle scale. PCA-CA identified at least two distinct regions within single particles. However, the spectral variations between these regions were much smaller than the variations of averaged NEXAFS spectra representing different particles from the same soil horizon, implying that interparticle heterogeneity is much larger than intraparticle heterogeneity. Especially the contents of aromatic and carboxyl carbon exhibited a large variability. Overall, the NEXAFS spectra of water-dispersible soil colloids were similar to the NEXAFS spectrum of the humic acid fraction, but differed clearly from the fulvic acid and dissolved organic matter fractions extracted from the same soil horizon using conventional techniques.     

Speciation of selenium in stream insects using X-ray absorption spectroscopy

Selenium contamination in the environment is a widespread problem affecting insects and other wildlife. Insects occupy a critical middle link and aid in trophic transfer of selenium in many terrestrial and freshwater food chains, but the mechanisms of selenium uptake through the food chain are poorly understood. In particular, biotransformation of selenium by insects into different chemical forms will greatly influence how toxic or benign the selenium is to that organism or to its predators. We have used X-ray absorption spectroscopy (XAS) to identify the chemical form of selenium in insects inhabiting selenium contaminated streams near Hinton, Alberta (Canada). Selenium K near-edge spectra indicate a variability of selenium speciation among the insects that included mayflies (Ephemeroptera), stoneflies (Plecoptera), caddisflies (Trichoptera), and craneflies (Diptera). Higher percentages of inorganic selenium were observed in primary consumers, detritivores, and filter feeders than in predatory insects. Among the organic forms of selenium, organic selenides constituted a major fraction in most organisms. A species modeled as trimethylselenonium was observed during the pupal stage of caddisflies. These results provide insights into how the insects cope with their toxic cargo, including how the selenium is biotransformed into less toxic forms and how it can be eliminated from the insects. More broadly, this study demonstrates the strengths of XAS to probe the effects of heavy elements at trace levels in insects from the field.     

Surface structure and composition of fresh and bloomed chocolate analysed using X-ray photoelectron spectroscopy, cryo-scanning electron microscopy and environmental scanning electron microscopy

Bloom on chocolate is an unsightly surface condition resulting in a white, powdery appearance that consumers interpret as a sign of poor quality. Bloom results from incorrect processing or storage. Two innovative methods have been developed, and used in conjunction, to investigate the morphology and composition of bloomed chocolate.Environmental scanning electron microscopy (ESEM) was used to image the morphology of the surface of fresh and bloomed chocolate and to observe the changes in morphology, in real time, during heating and cooling. The nodular morphology of sugar bloom was easily distinguished from the blade like crystals of fat bloom. Surface details for the fat blades indicated an extrusion mechanism during their formation on the chocolate surface.“Cold stage” X-Ray Photoelectron Spectroscopy (XPS) was used to analyse the surface composition of fresh and bloomed samples. The carbon species present in the bloom on poorly tempered chocolate were a combination of CH₂, C-OH, OC-OH and O-C-O groups indicating a mixture of sugar and fat bloom. Conversely the species present on bloomed, well tempered chocolate comprised only CH₂, C-OH, OC-OH indicating the bloom was comprised of fat (from cocoa butter) and no sugar.     

Speciation of Zn associated with diatoms using X-ray absorption spectroscopy

The long- and short-term interactions between zinc, an essential but also toxic element, and freshwater and marine diatoms are not well understood partly because of a lack of information on Zn speciation on the surface and inside the cells. In this work, interactions of aqueous Zn2+ with marine (Skeletonema costatum) and freshwater (Achnanthidium minutissimum, Navicula minima, and Melosira varians) diatoms were studied using conventional macroscopic techniques, while the local atomic structure of metal ions adsorbed on their surface or incorporated into the cells was characterized by in-situ Zn K-edge X-ray absorption fine structure (XAFS) spectroscopy on both intact and liophylized samples. At the cell surface for all diatom species studied, Zn was tetrahedrally coordinated with oxygen at approximately 2.00 +/- 0.02 A and monodentately bonded to one or two carboxylate groups; these results are consistent with the surface speciation model developed from macroscopic adsorption experiments. The atomic environment of Zn incorporated into freshwater diatoms during long-term growth in normal nutrient media was distinctly differentfrom that of adsorbed Zn: it was dominated by O (and/or N) neighbors in a tetrahedral arrangement at 1.97 +/- 0.02 A in the first atomic shell, with the presence of 1 phosphorus and 2 carbons in the Zn second shell. Contrasting speciation of intracellular zinc was revealed for the marine species Skeletonema costatum in which Zn was coordinated to 2 O/N atoms and 2 sulfur groups in the form of cysteine-histidine complexes and/or zinc thiolate clusters. These new structural data strongly suggest: (i) the predominant > R-COO- ligand binding of Zn atthe diatom surface; (ii) the nonspecific storage of Zn in the form of carboxylate/phosphate groups inside the cell of freshwater species; and (iii) the highly specific thiol-ligand coordination of intracellular zinc for marine S. costatum species.     

Three-dimensional mapping of oxygen distribution in wastewater biofilms using an automation system and microelectrodes

The three-dimensional oxygen distribution in wastewater biofilms was evaluated using combined oxygen microelectrodes and an automation system. The biofilms were sampled from rotating biological contactors treating domestic wastewater. The samples studied were mature biofilms with a thickness from 630 to 1600 microm. It was demonstrated that the dissolved oxygen concentration could be depleted at the biofilm surface. The heterogeneity of the dissolved oxygen distribution was high in sections further away from the biofilm surface in the water layer. The study showed that the concentration and level of heterogeneity of dissolved oxygen inside the biofilms decreased with depth, forming stratification. The oxygen concentration in biofilms changed generally from a high degree of heterogeneity near the biofilm surface to a low degree of heterogeneity in deep sections of biofilms, indicating a cell--cluster-like structure near the surface and more compact base layer close to the substratum. The three-dimensional oxygen distribution maps revealed pockets of dissolved oxygen in deep sections of biofilms. The dissolved oxygen concentrations of these pockets in the biofilm samples ranged from 0.4 to 1.0 mg/L at 760 microm depth. The three-dimensional oxygen distribution maps produced relevant knowledge of functional and structural characteristics of biofilms used for the treatment of wastewater.     

Speciation and characterization of arsenic in Ketza River mine tailings using X-ray absorption spectroscopy

Ketza River mine tailings deposited underwater and those exposed near the tailings impoundment contain approximately 4 wt % As. Column-leaching tests indicated the potential for high As releases from the tailings. The tailings are composed dominantly of iron oxyhydroxides, quartz, calcite, dolomite, muscovite, ferric arsenates, and calcium-iron arsenates. Arsenopyrite and pyrite are trace constituents. Chemical compositions of iron oxyhydroxide and arsenate minerals are highly variable. The XANES spectra indicate that arsenic occurs as As(V) in tailings, but air-drying prior to analysis may have oxidized lower-valent As. The EXAFS spectra indicate As-Fe distances of 3.35-3.36 A for the exposed tailings and 3.33-3.35 A for the saturated tailings with coordination numbers of 0.96-1.11 and 0.46-0.64, respectively. The As-Ca interatomic distances ranging from 4.15 to 4.18 A and the coordination numbers of 4.12-4.58 confirm the presence of calcium-iron arsenates in the tailings. These results suggest that ferric arsenates and inner-sphere corner sharing or bidentate-binuclear attachment of arsenate tetrahedra onto iron hydroxide octahedra are the dominant form of As in the tailings. EXAFS spectra indicate that the exposed tailings are richer in arsenate minerals whereas the saturated tailings are dominated by the iron oxyhydroxides, which could help explain the greater release of As from the exposed tailings during leaching tests. It is postulated that the dissolution of ferric arsenates during flow-through experiments caused the high As releases from both types of tailings. Arsenic tied to iron oxyhydroxides as adsorbed species are considered stable; however, iron oxyhydroxides having low Fe/As molar ratios may not be as stable. Continued As releases from the tailings are likely due to dissolution of both ferric and calcium-iron arsenates and desorption of As from high-As bearing iron oxyhydroxides during aging.     

Microstructure of milk gel and cheese curd observed using cryo scanning electron microscopy and confocal microscopy

Cryo scanning electron microscopy (cryo SEM) and confocal laser scanning microscopy (CLSM) were used to visualise changes in the microstructure of milk, rennet-induced gel and curd during the manufacture of Cheddar cheese. Our results show that cryo preservation did not alter the microstructure of the sample when it was fixed by rapid freezing in slush liquid nitrogen due to the formation of amorphous ice. Artefacts such as the formation of ice crystals could be observed in samples when immersed directly into liquid nitrogen (−196 °C) at atmospheric pressure. These ice crystals changed the shape of sample pores increasing their size to >20 μm. The etching time, thickness of gold coating, accelerating voltage and type of detector used for cryo SEM observation were varied in order to minimise the formation of such artefacts and optimise conditions for imaging. Chains and clusters of casein micelles and fat globules were best observed in the gel and the cooked curd when the samples were freeze fractured and etched for 30 min, coated with a mixture of gold and palladium alloy approximately 6 nm thick at −140 °C and observed using a backscattered electron detector at 15 kV. The structure of the gel, curd and cheese was also observed using CLSM. Spherical fat globules were mostly present in the serum pores of the gel prepared from unhomogenised milk but were found embedded in the aggregated chains of the casein network within the gel prepared from homogenised milk when observed using CLSM. The porosity measurements obtained using cryo SEM were similar to those obtained using CLSM. These two complementary techniques can potentially be used to assist studies for the control of cheese texture and functionality.     

Speciation of sulfate in size-fractionated aerosol particles using sulfur K-edge X-ray absorption near-edge structure

X-ray absorption near-edge structure (XANES) at sulfur K-edge was used to determine the sulfate species present in size-fractionated aerosol particles based on the postedge structure after the main absorption peak in the XANES region. A comparison of the XANES spectra of reference sulfate materials and aerosol samples collected in Tsukuba in Japan clearly showed that (NH4)2SO4 was the main sulfur species in particles with a smaller diameter and CaSO4 x 2H2O (gypsum) was the main sulfur species in particles with a larger diameter. A simulation of the XANES spectra by reference materials allows us to obtain the quantitative mixing ratios of the different sulfate species present in the aerosol samples. The presence of minor sulfur species other than (NH4)2SO4 and gypsum at the surface of mineral aerosols is suggested in our simulations and by a surface-sensitive conversion electron/He-ion yield XANES. In the absence of a contribution from a large dust event, the mole concentration of gypsum in the mineral aerosol fraction (particle diameter > 1 microm) determined by XANES is similar to that of Ca which is determined independently using ion chromatography. This shows that the Ca and sulfate in the mineral aerosols are present only as gypsum. Considering that calcite is the main Ca mineral in the original material arising from an arid and semiarid area in China, it is strongly suggested that gypsum is formed in aerosol during its long-range transportation by a reaction between calcite and sulfate ions.     

Compositions of maple sap microflora and collection system biofilms evaluated by scanning electron microscopy and denaturing gradient gel electrophoresis

The bacterial microflora of maple sap and biofilms in collection system tubing were studied through the use of bacterial counts, scanning electron microscopy (SEM) of surfaces and the analysis of 16S rRNA gene by denaturing gradient gel electrophoresis (DGGE). Samples were taken at five times during the 2002 and 2003 seasons in order to follow the changes in the microflora of this complex ecosystem. Bacterial counts showed the growth of bacterial populations as the season advanced. These populations were mainly composed of psychrotrophic bacteria and Pseudomonas spp. SEM results confirmed the suspected presence of biofilms on the inner surfaces of tubing samples. Bacterial colonization and biofilm formation progressively increased during the season for both lateral and main line surfaces, and biofilms were mainly composed of rod shape bacteria. The bacterial microflora profiles obtained for sap and corresponding biofilm by DGGE showed up to 12 major bands. The Shannon-Weaver index of diversity (H) calculated from DGGE bands were statistically higher for sap samples compared to biofilm. The diversity index was relatively stable or increasing for lateral line sap and biofilm samples during the season while the diversity index for sap and biofilm samples of the main line showed a decreasing profile as the season progressed. Sequence analysis of major DGGE bands revealed the predominance of bacteria from the genera Pseudomonas, Rahnella and another, unidentified genus. The results describe the composition of sap collection system microflora as well as the formation of biofilms and will be useful for further studies on factors affecting maple product quality.     

Confocal scanning laser microscopy and quantitative image analysis: application to cream cheese microstructure investigation

The naked eye observation of cream cheese confocal scanning laser microscopy images only provides qualitative information about its microstructure. Because those products are dense dairy gels, confocal scanning laser microscopy images of 2 different cream cheeses may appear close. Quantitative image analysis is then necessary to compensate for human eye deficiency (e.g., lack of precision, subjectivity). Two kinds of quantitative image analysis were performed in this study: high-order statistical methods and grayscale mathematical morphology. They were applied to study the microstructure of 3 different cream cheeses (same manufacturing process, same dry matter content, but different fat and protein contents). Advantages and drawbacks of both methods are reviewed. The way they may be used to describe cream cheese microstructure is also presented.     

Speciation of hepatic Zn in trout exposed to elevated waterborne Zn using X-ray absorption spectroscopy

The long-term impacts of chronic metal exposure for aquatic biota are not well understood, partly due to a lack of understanding of metal speciation within tissues. The objective of this study was to determine hepatic Zn speciation of rainbow trout (Oncorhnychus mykiss) exposed to Zn-enriched water in relation to unexposed (control) fish,through direct analysis of freeze-dried liver samples using synchrotron X-ray absorption spectroscopy (XAS). Juvenile rainbow trout (n=30) were exposed to Zn in a two-step process, 200 microg L(-1) for 14 days, followed by 370 microg L(-1) for 23 days. Thirty other trout were grown in a control treatment (10 microg Zn L(-1)). At the end of the experiment, three liver samples per treatment were collected, freeze-dried, ground, and mixed homogeneously. Although Zn concentration was higher in the Zn-exposed livers than in the control livers (22.32 vs 13.73 mg kg(-1), respectively; p < 0.05), Zn speciation was similar for both groups. Extended X-ray absorption fine structure (EXAFS) spectroscopy indicated that Zn was coordinated to 4 sulfur atoms with an average Zn-S bond distance of 2.31 +/- 0.02 A. Sulfur K-XANES analysis confirmed that S was predominantly in reduced organic form analogous to cysteine. Our results are consistent with previous evidence for Zn(II) bonding to S in metallothionein proteins. These results suggest that the mechanisms for dealing with the extra load of bioaccumulated Zn in high exposure conditions were the same as in the control group.     

Characterization of fibrillar collagen types using multi-dimensional multiphoton laser scanning microscopy

In dermal photodamage the ratio of the collagen types III to I changes. This makes the investigation of the fibrillar collagen type characteristics interesting for skin research. In this study collagen types were characterized using 5-dimensional multiphoton laser scanning microscopy (5D-IVT) that can be applied in vivo. Second harmonic generation (SHG) signals and fluorescence lifetimes of the collagen autofluorescence were analysed. Collagen type I generates a higher SHG intensity and a longer fluorescence lifetime compared to collagen type III. Thus, the SHG intensity decrease found in photodamaged skin might be explained by the increase in collagen type III. Calculating the in vivo relevant increase of collagen type III gives a negligible difference in fluorescence lifetime not qualifying this method for the determination of collagen type changes in dermal photodamage in vivo in human skin. However, for pathologies that exhibit higher differences in collagen types 5D-IVT analysis might be a suitable method.     

Distinguishing respirable quartz in coal fly ash using computer-controlled scanning electron microscopy

Determination and classification of quartz in coal fly ash (CFA) is a subject of interest because of the adverse health effects caused by inhalation of crystalline silica. Workers with prolonged exposure to this carcinogen can develop respiratory diseases over time. This obviously may include utility plant workers involved in the handling, loading, and hauling of CFA. In this investigation, computer-controlled scanning electron microscopy (CCSEM) and X-ray diffraction (XRD) were used to investigate Si-rich phases in CFA to develop a better approach for the determination of respirable quartz. Three CFA samples from utility boilers and a NIST standard CFA sample were investigated. The XRD measurements indicated that the four samples contained from 7.0 to 16.0 wt.% of quartz. The CCSEM measurements utilized both particle size distributions and a particle shape parameter, circularity, to classify the Si-rich phases in these ashes as either crystalline or amorphous (glass). The results indicated that the amount of free, respirable, quartz in these CFA samples ranged from only 0.1-1.0 vol % and showed little correlation with the XRD results for the bulk ash. These results are significant in view of the factthat XRD is the traditional method of measuring crystalline silica in dust collected from workplace atmospheres.     

Direct observation of bacterial exopolysaccharides in dairy products using confocal scanning laser microscopy

The objective of this work was to develop a methodology for direct visualization of bacterial exopolysaccharides (EPS) in fully hydrated dairy products. The new method involved staining EPS with wheat germ agglutinin labeled with Alexa fluor 488 or staining with concanavalin A 488. Samples were observed using confocal scanning laser microscopy. Distribution of EPS produced by Lactococcus lactis (CHCC 3367), a combination of Streptococcus thermophilus (CHCC 3534) and Lactobacillus delbrueckii ssp. bulgaricus (CHCC 769) and Lactobacillus delbrueckii ssp. bulgaricus RR in milk was compared in stirred and unstirred fermented milk. The EPS and proteins were observed as distinct entities, with EPS present in the protein network pores. EPS was observed in greater amounts in milk fermented by the ropy L. lactis culture than in milk fermented by the less ropy strain of S. thermophilus. Stirring the fermented milk caused aggregation of EPS into more extended structures. The more ropy the culture, the longer and larger the strands formed during stirring. The method was also applied to Feta cheese made with an EPS-producing strain of S. thermophilus. EPS was observed in the cheese as thick sheets filling pores in the protein network.     

Three-dimensional imaging and analysis of the surface of hair fibres using scanning electron microscopy

Cuticle scales are the most obvious feature of a hair fibre's outer surface and much research has focused on assessing the influence of surface topography on the associated hair fibre's properties. However, much of the research has either been qualitative or, if quantitative, employed relatively laborious analytical techniques to establish the necessary statistical robustness. In this study, we report on the application of a 3D image analysis package capable of producing 3D data from multiple 2D scanning electron microscope (SEM) images of hair fibres. Analysis of the surface profile can be carried out quickly and accurately, enabling quantification of the scale structure. To validate the novel technique and ensure that the scale heights measured were indeed accurate and reproducible, extensive calibration of the SEM and the 3d software has been performed. In addition, scale heights on a single hair have been determined by using atomic force microscopy (AFM) and the results compared with analogous data produced from the same scale edges using the 3D image analysis technique. The data obtained indicate that a relatively quick, accurate and viable method to determine scale height in keratin fibres has been established. A further 3D-SEM analysis method has been developed which allows individual scales to be monitored and for the cuticular scale height to be quantified after repeated 'smoothing' treatments.     

Evaluating variability and uncertainty separately in microbial quantitative risk assessment using two R packages

Quantitative risk assessment has emerged as a valuable tool to enhance the scientific basis of regulatory decisions in the food safety domain. This article introduces the use of two new computing resources (R packages) specifically developed to help risk assessors in their projects. The first package, “fitdistrplus”, gathers tools for choosing and fitting a parametric univariate distribution to a given dataset. The data may be continuous or discrete. Continuous data may be right-, left- or interval-censored as is frequently obtained with analytical methods, with the possibility of various censoring thresholds within the dataset. Bootstrap procedures then allow the assessor to evaluate and model the uncertainty around the parameters and to transfer this information into a quantitative risk assessment model. The second package, “mc2d”, helps to build and study two dimensional (or second-order) Monte-Carlo simulations in which the estimation of variability and uncertainty in the risk estimates is separated. This package easily allows the transfer of separated variability and uncertainty along a chain of conditional mathematical and probabilistic models. The usefulness of these packages is illustrated through a risk assessment of hemolytic and uremic syndrome in children linked to the presence of Escherichia coli O157:H7 in ground beef. These R packages are freely available at the Comprehensive R Archive Network (cran.r-project.org).     

Identification of cheese mite species inoculated on Mimolette and Milbenkase cheese through cryogenic scanning electron microscopy

Samples of Mimolette (France) and Milbenkase (Germany) cheeses traditionally ripened by mites were analyzed to determine the mite species present on each sample. Scientific literature was reviewed to understand which mite species most commonly infest cheese. Morphological features possessed by mites were then studied to understand what unique characteristics are required to ensure accurate identification. After identification and compilation of a detailed key of stored food mites (subclass Acari, order Astigmata) and their delineating features, the mites were viewed through a cryogenic scanning electron microscope. It was determined that Mimolette cheese is inoculated with Acarus siro L. The features studied to identify this mite species included idiosomal length and shape, setae length and arrangement, leg size, placement of anus and genitals, and solenidia shape. The Milbenkase cheese is inoculated with Tyrolichus casei Oudemans, which was evident after viewing the same features used to identify A. siro and the supracoxal seta shape. With this knowledge, further research can be conducted on the 2 cheese varieties to understand what chemical, physical, and microbial changes occur within the cheeses because of mites. It is important to identify the mite species present on each cheese variety to improve our understanding of their role in creating the distinctive characteristics that set these cheeses apart from others.