Sticky Wax Infiltration in the Preparation of Sawed Undecalcified Bone Sections

The undecalcified bone specimen was surfaced by an ordinary motor-driven circular saw. After thorough drying in air, the specimen was infiltrated with melted Caulk sticky wax (L. D. Caulk Co., Milford, Del., 19963) without casting in a block. The specimen was affixed to the Gillings-Hamco thin-sectioning machine with cut surface parallel to the circular diamond blade. Prior to sawing each section, the specimen surface was blown dry and coated with a thin supporting layer of stick wax. The section was then attached to an albumen-coated glass slide with the newly cut surface facing the slide. After drying in room temperature, the slide was soaked in xylene to partially dissolve the sticky wax, and the loosened residue was removed subsequently by gentle brushing. The section was mounted and covered with a coverglass. Sections 50-100 μ thick were prepared satisfactorily by this method. The advantages of using sticky wax as an infiltration medium depend on its physical properties: it is gluey when melted, and holds the bony trabeculae together; it becomes hard and nonsticky at room tempperature, and can be sawed together with bone tissue. Since a new layer of wax blends readily with the old wax surface, it allows the important supportive coating of wax to be added to the sawing surface for each section cutting     

REMOVING GOLD COATING FROM SEM SAMPLES

Abstract:  An essential tool in micropalaeontological studies is analysis and imaging using a scanning electron microscope (SEM). It is commonly necessary to coat the sample with a thin layer of gold or gold-palladium alloy in order to prevent charging of the surface, to promote the emission of secondary electrons so that the specimen conducts evenly, and to provide a homogeneous surface for analysis and imaging. However, coating a specimen with gold is a semi-destructive process that masks the surface of specimens, and a common curatorial practice is to prohibit coating of important specimens. We describe a non-destructive, simple and inexpensive technique that uses KCN (or NaCN), a glass jar, paper towels, cardboard and a fume hood for the removal of gold coatings from SEM samples. This technique requires little additional handling of the specimen, and there is no need to place the specimen in a solution.     

Interfragmentary surface area as an index of comminution energy: proof of concept in a bone fracture surrogate

Fracture mechanics theory postulates a monotonic relationship between energy absorption and fracture surface generation. We hypothesized that this relationship was demonstrable to the point that, on a continuous scale, comminuted fractures created with disparate levels of energy delivery could be discriminated. Using a bone fracture surrogate in conjunction with digital image analysis of CT fracture data, we measured the surface area freed by controlled, discrete fracture simulations. Prior to these simulations, the reproducibility of the digital image analysis algorithm was validated with repeated measurements by two different operators. The parametric fracture series results showed a statistically significant difference in measured de novo surface area between four specimen groups, over a range of input energies from 1.4 x 10(10)-9.1 x 10(10)J/m(3) (or 12.5-80.2J/specimen). The results of this study provide confirmation that comminution severity can indeed be measured on a continuous scale, based on energy absorption (another clinically meaningful index).     

Using Scrapings from Formalin-Fixed Tissues to Diagnose Leptospirosis by Fluorescent-Antibody Techniques

Small specimens of formalin-fixed tissues approximatey 1 × 1 × 0.2 cm were cut from the suspect specimen. Several clean microscope slides were dipped in 1% aqueous gelatin and air-dried or dried on a slide warmer. Each tissue specimen was washed in running tap water for 2-5 min and then lightly scraped with a straight knife blade, cutting edge perpendicular to the surface of the specimen. The scrapings were allowed to build up and cling to the knife blade, which was then turned so that the broad surface contacted the slide; thus, the scrapings could be smeared onto the slide in a single motion. Sufficient pressure was applied to embed the tissue fragments in the gelatin coating. Smears, dried in air or on a slide warmer, were stained immediately by a standard direct or indirect technique to detect fluorescein-labeled antigens. This scraping method, adapted to the study of leptospirosis by fluorescent-antibody technique, could reduce the need for cryostat-cut tissues and facilitate the observation of individual leptospires.     

The effect of specimen geometry on the mechanical behaviour of trabecular bone specimens.

The effect of specimen geometry on the mechanical behaviour of trabecular bone specimens was studied by non-destructive uniaxial compression to 0.4% strain using cylindrical specimens with different sizes and length-to-diameter ratios, and by comparing cubic and cylindrical specimens with the same cross-sectional area. Both the length and the cross-sectional area of the specimen had a highly significant influence on the mechanical behaviour (p less than 0.0001). Within the actual range of length (2.75-11.0 mm) the normalized stiffness (Young's modulus) was related nearly linearly to the specimen length. This dependency on specimen length is suggested to be caused mainly by structural disintegrity of the trabecular specimens near the surface. The normalized stiffness (Young's modulus) was also positively correlated to the cross-sectional area. This dependency on cross-sectional area is probably due to friction-induced stress inhomogeneity at the platen-specimen interface. A cube with side length 6.5 mm or a cylindrical specimen with 7.5 mm diameter and 6.5 mm length are suggested as standard specimens for comparative studies on trabecular bone mechanics.     

Nano- and Micro-Scale Surface Modification of FCC Metal Using High Submerged Cavitating Water Jet

The aim of this paper is to establish a possible application of the cavitation phenomenon as an efficient method to modify surface properties at the nano and micro levels. Commercial-purity copper was subjected to high submerged cavitating jets under different initial conditions, for time periods between 15 and 1,800 s. The force generated by jet cavitation is employed to modify the surface roughness in the order of nano and micro scales. The target surface was analyzed with optical as well as scanning probe electron microscopy. The results showed the possibility to use cavitation bubbles to establish a nanofabrication method for the surface preparation, shoot-less surface peening (nano/micro level). Also, the cavitation is assumed to be one of the miniaturized testing methods that have to be developed to reliably measure mechanical properties in small dimensions and to identify the behavior caused by the size dependence. With optical, SEM, and atomic force microscopy observation techniques in this study, the deformation mechanism and the formation of planar or wavy slip were also studied. The results indicate that even at short exposure times, observed roughness having a characteristic “serpentine” configuration can be related to the start of the plastic deformation of the specimen surface. Longer exposure times inevitably result in a greater number of jet–specimen interactions leading to specimen fracture.     

Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy

The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.     

Micro-plasma textured Ti-implant surfaces

The surface state of titanium implants modulates bone response and implant anchorage. This evidence brought implant manufacturers to switch from the standard surface refinements and implement new surface treatments for more bone apposition and enhanced interfacial strength measured by removal torque or push-out tests. Anodic plasma-chemical treatment of implant surfaces is a cost-effective process to modify surface topography and chemistry. This technique is used for structuring connected with a coating of implant surfaces. The aim of our investigations, here, is to texture the implant surface in the nanoscale without coating. Ti disks with different mechanical pre-treatment (grinded, glass blasted) were used as substrate. Micro-plasma texturing was carried out in an aqueous electrolyte. By applying a pulsed DC voltage to the specimen, micro-plasma discharge was generated in the thin steam film between immersed specimen and electrolyte. The electrical process parameter current density was varied. The micro-plasma textured Ti surfaces were characterised optically by SEM and electrochemically by CV- (for testing the corrosion parameters), CA- (to give the enlargement of the real surface) and EIS-measurement in range of 100 kHz-100 microHz. We found that the initial structure of the material surface has small or no influence on the results of the micro-plasma treatment. The properties of the thick oxide layer resulting from the plasma process are influenced by electrical process parameters. After removal of the thick oxide layer a fine, micro- and nanoscaled surface structure of the titanium remains.     

Pipette aspiration technique for the measurement of nonlinear and anisotropic mechanical properties of blood vessel walls under biaxial stretch

A pipette aspiration technique was proposed for the measurement of nonlinear mechanical properties of arteries under biaxial stretching. A cross-shaped specimen of porcine thoracic aorta whose principal axes corresponded with the axial and circumferential directions of the aortic walls was excised. The intraluminal surface of the specimen was aspirated with a circular cross-sectioned glass pipette while the specimen was stretching in the axial and circumferential directions in 10% increments. The elastic modulus agreed with the incremental elastic modulus obtained through a conventional pressure-diameter test of the same specimen to within an error of 30% at a circumferential stretch ratio below 1.3 and an axial stretch ratio of 1.0, 1.1 or 1.2, which represent lower range of physiological stretch ratios for the porcine aorta. A rectangular cross-sectioned pipette was utilized to measure anisotropic properties of the specimen under biaxial stretching. When aspirated with such a pipette, the specimens' elastic properties along the length of the rectangular pipette cross section can be neglected. The elastic modulus was found to increase rapidly when the specimen was stretched in the direction of the pipette's width. Thus, pipette aspiration should have many advantages such as well measurement of the local nonlinear and anisotropic mechanical properties of blood vessel walls.     

Hot Melt “Corner Point Method” for Attaching Large Plastic Sections to Glass Slides

We describe a fast method for firm attachment of large plastic sections to glass slides with EVA-copolymers, commonly known as hot melt sticks. Solid hot melt sticks dissolve slowly in xylene to form an adhesive gel within 6 hours. Small drops of hot melt gel are applied to the corners of the sections and surrounding slide surface at ambient or elevated temperatures. The gel sticks to both the plastic and the glass slides. The hot melt “corner point method” prevented detachment of sections in staining procedures. As an additional technique, we suggest the use of hot melt adhesive for attaching plastic specimen blocks to wooden blocks or metallic specimen holders.     

Method of non-destructive mechanical testing of new surface coatings for prostheses]

Research into new surface coatings and surface processing methods for prostheses is subject to numerous studies. The aim of this study was to test an innovative biomechanical measuring method for the examination of the ingrowth of bone implants. Using a transcortical model, coated (n=14) or uncoated (n=14) titanic cylinders were implanted into the lateral condyle of 28 New Zealand White Rabbits. After 6 weeks or 6 months the animals were sacrificed and the osseointegration of the implants was evaluated biomechanically and histologically. Up to traction of 50 N the load dependent movement between bone and testing cylinder did not lead to a destruction of the bone-implant-interface. Therefore, biomechanical and histological investigations could be performed in the same specimen. The results of both evaluations showed a significant correlation (correlation coefficient -0.79; p < 0.01) and were absolutely reproducible. With the method of non-destructive mechanical testing, it is possible to halve the number of required animals. Additionally, the results of the biomechanical and histological analysis can be compared and thus serve as an internal control. In summary, the method of non-destructive mechanical testing represents an ideal tool to study new surface coatings and surface processing methods for prostheses.     

Mechanical and failure behaviour of the stratum corneum

The load-deformation-time behaviour of heat-separated human stratum corneum was investigated using a pure shear specimen geometry. The tissue displayed non-linear load-deformation behaviour and stress relaxation, although the extensibility and amount of stress relaxation was considerably less than that shown by other soft connective tissues. Controlled failure tests were carried out after an edge cut had been made in the pure shear specimen. Sources of secondary failure, either at the free edge of the specimen, or due to the presence of inhomogeneities in the tissues, were common. Analysis of the test results suggested that the fracture surface energy of stratum corneum has a mean value of 3.6 kJ m-2 which is comparable with the tougher synthetic polymers.     

Wing colors of Chrysozephyrus butterflies (Lepidoptera; Lycaenidae): ultraviolet reflection by males

Wing colors of the four species of Chrysozephyrus butterflies were analyzed by a spectrophotometer. As the dorsal wing surface of males showed a strong reflectance when the specimen was tilted, measurements were made by the tilting method. The dorsal wing surface of males which appears green to the human eye reflected UV (315-350 nm) as well as green light (530-550 nm). The reflectance rate of UV to visible green light varied among species with a higher rate for C. hisamatsusanus and C. ataxus, and a lower rate for C. smaragdinus and C. brillantinus. The peak wavelength and the peak height did not shift when the specimen was exposed to direct sunlight at least for 16 hr. Artificial removal of scales by scratching the wing surface decreased reflectance. Blue marks on the forewings of C. brillantinus, C. hisamatsusanus and C. ataxus females reflected UV to visible light of short wavelength, and orange marks on the dorsal surface of the forewing and the ventral surface of the hindwing of C. samaragdinus females showed a higher reflectance at longer wavelengths.     

THE NEAR-SPINELESS TRACHELOMONAS GRANDIS (EUGLENOPHYCEAE) SUPERFICIALLY APPEARS SPINY BY ATTRACTING BACTERIA TO ITS SURFACE1,2

Trachelomonas grandis Singh has a mucilaginous, highly porous mineralized lorica (envelope) generally without ornamentation except occasionally for a few short, tapered, anterior or posterior spines. However, as our first cultures of this species aged, rod-shaped structures appeared on the loricas. That these surface projections were bacteria was determined by scanning and transmission electron microscopy. The bacteria, 2-6 μm long with rounded apices, were oriented perpendicular to the exterior lorica surface and were attached on one end by apically produced tie-down fibers. The bacteria also secreted fibers over their entire surface, forming a network between them that collapsed during specimen preparation for scanning electron microscopy. The density of the surface bacteria increased with time until the lorica took on a “spiny” appearance superficially similar to lorica extensions of algal origin. In mature algal specimens, an estimated 1200-1800 bacteria per lorica occurred as a monolayer, the maximum number related to the surface area of the lorica available for bacterial colonization. The bacteria, always motionless while attached, showed putative evidence of budding. Fission formed short chains of up to three cells on the lorica. Our cultures maintained this association for 8 years (1972-1979). However, cultures ordered for further study in the past year have failed to develop loricas with more than just a few bacterial cells, and most have none.     

A gracile hominid cranium from upper member G of the Shungura Formation,Ethiopia

A fragmentary hominid cranium with teeth, specimen L.894-1, dating from 1.84 m.y. BP in the Shungura Formation at Omo, is described. From its dental and cranial morphology and because of similarities to Olduvai Hominids 24 and 13 and Sangiran 4, among others, it is concluded that the specimen represents a member of an early species of the genus Homo (Homo habilis or Homo modjokertensis). The specimen shows approximal grooving on the premolars, pre-mortem chipping of the molar enamel, foramina ovale and spinosum divided by the sphenosquamosal suture, limited pneumatization of the mastoid region, and a possible interparietal bone. Sedimentological, ostracod, pollen, macrofloral, and taphonomic data indicate that the paleo-environmental context was a savanna/grassland or savanna woodland on the margin of a saline lake.     

Distortion-Free Mounting of Frozen Sections by Handling on Paper Supports

Frozen sections are cut from the specimen until the level of interest is reached. A strip of paper (bond or similar writing paper) 5 cm long and about 1 cm wider than the specimen is moistened with water, closely applied to the surface of the specimen and frozen onto it. As the section is cut, the end of the paper strip above the knife is grasped and turned backward toward the other end of the strip. The section is then applied to an albumenized glass slide, firmed and thawed by finger pressure, and the paper removed. After thorough drying, the preparation is ready for further processing. When properly performed, mounted sections whose details coincide to those of the uncut block can be obtained. If thawing on the knife is prevented by cooling the knife, the technic can be performed without a cryostat, but it is also feasible to use a cryostat if a favorable temperature is maintained. The authors obtained 30 μ serial sections, suitable for stereotaxic mapping, from rabbit brain by this method.     

Clevelandodendron ohioensis,gen. et sp. nov., a slender upright lycopsid from the late Devonian Cleveland Shale of Ohio

Clevelandodendron ohioensis Chitaley & Pigg gen. et sp. nov. is an almost entire lycopsid plant known from a single compressed specimen from the Cleveland Shale member of the Upper Devonian Ohio Shale. This unique specimen is 125 cm long, consisting of an unbranched, slender, monopodial axis with a partially preserved plant base bearing thick appendages at one end, and a compact, terminal ovoid bisporangiate strobilus at the other. The stem is 2 cm wide for most of its length. Visible on the decorticated stem surface are helically arranged, elongate leaf traces and laterally compressed, slender leaves along the stem margin. The plant base bears 4-6 thick appendages. The terminal strobilus is compact, ovoid, 9 cm long and up to 6 cm wide, morphologically similar to those of some Lepidodendrales, and bears helically arranged sporophyll/sporangium complexes with narrow bases and distal laminae up to 18 mm long, turned upward. Megaspores are 320-360 μm, trilete and laevigate, lacking a gula; microspores are 30-42 μm, trilete, indistinctly punctate and possibly assignable to Calamospora or Punctatisporites. Clevelandodendron demonstrates that slender unbranched lycopsids with an isoetalean plant habit similar to the Carboniferous genera Chaloneria and Sporangiostrobus and Triassic Pleuromeia-like forms were present as early as the Late Devonian. The early occurrence of this unique habit suggests that diversification within the isoetalean clade sensu Rothwell and Erwin (including both Isoetales and Lepidodendrales) was well established prior to the Carboniferous.     

Hydration force in the atomic force microscope: A computational study.

Using a hard sphere model and numerical calculations, the effect of the hydration force between a conical tip and a flat surface in the atomic force microscope (AFM) is examined. The numerical results show that the hydration force remains oscillatory, even down to a tip apex of a single water molecule, but its lateral extent is limited to a size of a few water molecules. In general, the contribution of the hydration force is relatively small, but, given the small imaging force ( approximately 0.1 nN) typically used for biological specimens, a layer of water molecules is likely to remain "bound" to the specimen surface. This water layer, between the tip and specimen, could act as a "lubricant" to reduce lateral force, and thus could be one of the reasons for the remarkably high resolution achieved with contact-mode AFM. To disrupt this layer, and to have a true tip-sample contact, a probe force of several nanonewtons would be required. The numerical results also show that the ultimate apex of the tip will determine the magnitude of the hydration force, but that the averaged hydration pressure is independent of the radius of curvature. This latter conclusion suggests that there should be no penalty for the use of sharper tips if hydration force is the dominant interaction between the tip and the specimen, which might be realizable under certain conditions. Furthermore, the calculated hydration energy near the specimen surface compares well with experimentally determined values with an atomic force microscope, providing further support to the validity of these calculations.     

Rosselia socialis: a dwelling structure of a probable terebellid polychaete

The trace fossil Rosselia socialis Dahmer 1937 is a funnel-shaped structure characterized by a concentrically laminated wall having a narrow cylindrical shaft at the central portion. In association with well-preserved funnel-shaped R. socialis , many spindle-shaped R. socialis -like specimens are abundant in the Middle Pleistocene Kongochi Formation in central Japan. While the former always occur just below an erosional bedding plane or outcrop surface parallel to the bedding, the latter are found in sediment showing no breaks in sedimentation. This suggests that the funnel-shaped 'R. socialis' is actually the truncated lower part of the spindle-shaped structure. In one specimen, deformation of laminae appears to have formed synchronously with construction of the wall. Morphology of the structure strongly suggests that the wall of R. socialis was formed through outward expansion caused by accretion of muddy material at the inner surface of the wall by the trace-maker inhabiting the central burrow. A specimen that was covered by a volcanic ash has the wall made up of the ash. This indicates that the maker of the burrow utilized surface sediment to construct the wall; that is, the trace-maker was possibly a detritus feeder. The terebellid polychaetes are reasonable candidates for the trace-makers. □ R osselia socialis , Japan, Middle Pleistocene, trace fossil, trace-making process, trace-maker.