Monitoring of the environment for asbestiform minerals has recently been of major interest, and analysis methods for the samples have been variable in reliability. Because of the difficulty in preparing suitable standards, acceptable criteria for reproducibility in asbestos analytical techniques have been that results on the same sample should agree within a factor of two or three. Various analytical techniques have been reported, but most of these rely on complete dissolution or oxidation of the filter medium used for collection and preparation of a transmission electron microscope sample. One technique reported uses ultrasonic treatment of the membrane in distilled water to remove fibers, after which the filter is discarded. Unfortunately, there is then no record of how many fibers remained attached and were discarded along with the filter membrane.

The use of stereoscopy to characterize three-dimensional structures observed by TEM has become widespread since the introduction of instruments operating at 1 MV. In its emphasis on whole structures and thick specimens this approach differs significantly from conventional methods of microstructural analysis based on three-dimensional image reconstruction from a number of thin-section views. The great advantage of stereo derives from the ability to directly perceive and measure structures in three-dimensions by capitalizing on the unsurpassed human ability for stereoscopic matching of corresponding details on picture pairs showing the same features from different viewpoints. At this time, stereo methods are aimed mainly at structural understanding at the level of dislocations, precipitates and irradiation-induced point-defect clusters in crystal and on the cellular level of biological specimens. 3-d reconstruction methods have concentrated on the molecular level where image resolution requirements dictate the use of very thin specimens.

For the most part the stereo methods used in TEM are direct and unsophisticated. Stereo pairs of micrographs are taken by tilting the specimen to record two views of the same structural features from viewpoints ten or so degrees apart.

Scanning electron microscopy studies of human hair defects have been previously confined to descriptions of variation in cuticle morphology and general appearance of the shaft. While the general structure of the hair shaft, i. e., nodes, longitudinal invaginations and twists, occur in regular patterns for given diagnostic categories, there is some question as to the validity of using cuticle morphology as a diagnostic index, since variation within a given subject is usually greater than between subjects. Also, there is a limited amount of information to be obtained from such studies since the metabolically active part of the structure is not visualized. Consequently, the present investigation was initiated to delineate the root sheath through which the emerging hair shaft travels.

The hair of Monilethrix is characterized by a noded appearance with the internode being very susceptible to breakage resulting in hair loss at the scalp. Freshly epilated hairs from a moderately affected male Caucasian having light brown hair were used.

Histological examination of the bulb indicates the presence of vacuoles in the cortical prekeratinized area of the zone of cellular differentiation. Fig. 1 shows a morphological defect in the cells of the precortical region of the germinal matrix. The nuclei of several of these cells have become elongated and in some cases, string-like, with the nuclei becoming pyknotic. It is possible that the lack of a functioning nucleus early in cell growth results in large open areas or vacuoles. Gross structural analysis indicates that the internode region of the shaft has less cortical area than does the node.

Pseudofolliculitis barbae (PF) is a dermatological disorder affecting approximately 50%. of black men who shave. The disease is characterized by ingrown hairs and attendant papules, pustules or vesicles ("razor bumps") which can occur over the entire beard area but which are most prevalent in the submandibular region. While it is generally accepted that the disease is of biological origin, PF appears to be triggered by shaving which in turn leads to the immediate cause of the disorder, ingrown hair formation. However, differences of opinion exist as to the basic etiology of PF--that is, how ingrown hair formation occurs in certain individuals and not others. Therefore a comparison of the skin, hair and post-shave beard hair regrowth patterns of "normal" and PF-prone black and white men by scanning electron microscopic (SEM) examination of polyethylene replicas was undertaken to determine the causes of ingrown hair formation.

Our studies of mammary tumor virus have included the application of the unlabeled antibody enzyme method of Sternberger to mammary tumor derived mouse cells in culture and observation with an electron microscope. The method avoids the extravagance of covalent binding of indicator molecules (horseradish peroxidase) with precious antibody locator molecules by relying instead upon specific antibody-antigen linkages. Our reagents included: Primary Antibody, rabbit anti-murine mammary tumor virus (MuMTV) which was antiserum 113 AV-2; Secondary Antibody, goat anti-rabbit IgG gamma chain (Cappel Laboratories); andthe Indicator, rabbit anti-horseradish peroxidase - horseradish peroxidase complex (PAP) (Cappel Labs.). Dilutions and washes were made in 0.05 M Tris 0.15 M saline buffered to pH 7.4. Cell monolayers, after light fixation in glutaraldehyde, were incubated in place by a protocol adapted from Sternberger and Graham and Karnovsky, then embedded by our usual method for monolayers. Reagents were confined to specific areas by neoprene 0-rings (Parker Seal Co.) reducing the amount of reagent needed to 50 microliters, 1/6th of that required to wet a 35 mm petri dish.

One of the problems in the study of mammary carcinomas is the cellular heterogeneity which they present. The use of a specific histochemical marker can help to distinguish the two principal cellular types found in the mammary gland; the epithelial and myoepithelial cells.

Mammary glands of female Balb/C mice of different ages were removed and fixed in 1.5% glutaraldehyde in 0.1M cacodylate buffer pH 7.2 with 1% sucrose. After two hours of fixation at 4°C, the material was washed in 0.1M cacodylate buffer, sectioned with a Smith-Farquar microtome set at 70-100u, and incubated for the histochemical detection of ATPases. The tissue was then washed again with 0.1M cacodylate buffer, postfixed in 1% osmium tetroxide, dehydrated, and embedded in an Epon-Araldite mixture.

For the Mg++ dependent ATPase the conventional method of Wachstein and Meisel was followed. The Mg++ dependent ATPase is localized in the plasma membranes of the epithelial and myoepithelial cells (Fig. 1).

This cytochemical study was designed to investigate early metabolic changes in the aortic wall that might lead to or accompany development of atherosclerotic plaques in rabbits. The hypothesis that the primary cellular alteration leading to plaque formation might be due to changes in either carbohydrate or lipid metabolism led to histochemical studies that showed elevation of G-6-Pase in atherosclerotic plaques of rabbit aorta. This observation initiated the present investigation to determine how early in plaque formation and in which cells this change could be observed.

Male New Zealand white rabbits of approximately 2000 kg consumed normal diets or diets containing 0.25 or 1.0 gm of cholesterol per day for 10, 50 and 90 days. Aortas were injected jin situ with glutaraldehyde fixative and dissected out. The plaques were identified, isolated, minced and fixed for not more than 10 minutes. Incubation and postfixation proceeded as described by Leskes and co-workers.

Several types of mammalian sensory receptor cells possess modified cilia which may play a primary role in stimulus detection. Axonemes and basal apparatus from other types of cilia and flagella have been isolated, characterized and compared in detail. In this communication we report results of what may be the first attempt to isolate and characterize such structures from a sensory cilium, the bovine retinal rod outer segment.

Bovine eyes were obtained from a nearby abattoir, the retinas were removed and swirled vigorously in a cold solution of 50% sucrose, 5 mM Tris, 2 mM MgCl2, pH 7.5, to detach outer segments. The suspension was then filtered through cheesecloth and the filtrate was centrifuged at 20,000 rpm for 1 hr. Rod outer segments were collected from the solution surface, diluted, layered onto a continuous sucrose gradient (25-38% sucrose in Tris MgCl2 buffer) and centrifuged for 3-12 hrs. at 10,000 rpm.

The initiation of association between cultured soybean root cells and Rhizobia shows specificity under proper conditions of culture. The establishment of these culture conditions for symbiotic association have been described previously. The compatible plant cells produced a filamentous extracellular material which was capable of trapping Rhizobia. Non-compatible cells did not produce extracellular material and were not invaded. In this presentation we have followed the effect of Rhizobial invasion on the wall morphology of the soybean root cells in suspension cultures. Changes in the morphology of the soybean cell walls were then examined as follows.

Harosoy root cells were grown in liquid culture in Gamborg's B-5 medium for two weeks. Rhizobium japonicum strain 138 was added to the culture for another 3 days. For this purpose, fixation was carried out by adding glutaraldehyde to the medium to a final concentration of 2%. The cell clumps were fixed 1 hr.

Ever since the introduction of glutaraldehyde as a fixative in electron microscopy of biological specimens, the identification of impurities and consequently their effects on biologic ultrastructure have been under investigation. Several reports postulate that the impurities of glutaraldehyde, used as a fixative, are glutaric acid, glutaraldehyde polymer, acrolein and glutaraldoxime.

Analysis of commercially available biological or technical grade glutaraldehyde revealed two major impurity components, none of which has been reported. The first compound is a colorless, water-soluble liquid with a boiling point of 42°C at 16 mm. Utilizing Nuclear Magnetic Resonance (NMR) spectroscopic analysis, this compound has been identified to be — dihydro-2-ethoxy 2H-pyran. This impurity component of the glutaraldehyde biological or technical grades has an UV absorption peak at 235nm. The second compound is a white amorphous solid which is insoluble in water and has a melting point of 80-82°C. Initial chemical analysis indicates that this compound is an aldol condensation product(s) of glutaraldehyde.

A new, variable phase contrast microscope, the PolanretTM, has been developed which is particularly suited to the optical screening of thin sections for electron microscopy with regard to tissue orientation and section quality.

By preliminary examination, defects in preparation of the specimen can be discovered immediately after sectioning and prior to staining or examination with the electron microscope. Such examination of unprocessed sections which are thinner than conventional light microscopy sections is possible because the Polanret TM system can make exceptionally small differences in optical path. Using the PolanretTM microscope, the phase and amplitude can be adjusted continuously so as to achieve optimum specimen contrast. The microscope can thus be used to visualize specimen defects and intracellular details in a way which conventional phase microscopy is unable to do.

This preliminary screening technique was evaluated for its usefulness in detecting microtome knife defects, i.e., scratches and chatter, so that corrective measures could be taken.