A Technical Improvement for Sectioning Hard Laminated Fibrous Tissues for Electron Microscopic Studies

The organization of the fibers in tissues of laminated fibrous structures constitutes an obstacle to obtaining ultrathin sections for transmission electron microscopic observations routinely. This paper deals with the orientation of the specimen block face in a way that greatly improves sectioning of these tissues.     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

A Structural Study of Isolated Mammalian Centrioles Using Negative Staining Electron Microscopy

We have used a combination of centrifugation onto electron microscope grids and negative staining to study the structure of isolated mammalian centrioles. The technique relies on visualisation of structural detail by use of a goldthioglucose negative stain. The approach provides an easy structural definition of the mature and immature centriole and has revealed some novel proximal projections on the mature centriole. The rapid technique should prove of use in future analyses of centriolar structure and biochemistry.     

A New Imprinting Material for Scanning Electron Microscopic Studies

A new imprinting material, “Ther-mocool”, has been used for preparing negative replicas of plant as well as metal surfaces for SEM study. The technique has wide application and gives very good results.     

Intraphagocytic Degradation of Group A Streptococci: Electron Microscopic Studies

The morphological changes that occur during intraphagocytic digestion of group A streptococci were studied by electron microscopy The first evidence of degradation of the ingested organism was the appearance of reticular changes in the bacterial endoplasm. This was followed by gradual swelling and dissolution of the bacterial wall, with final degradation of all the constitutuents to electron-dense debris. Accompanying changes in the phagocytic cell were observed; they consisted of vacuole formation, fusion of lysosomes with the wall of the yacuole, release of the lysosomal contents into the vacuole, and aggregation of the lysosomal contents around the ingested organism. Changes in the morphology of the organism similar to those observed during intraphagocytic digestion were also obtained by subjecting streptococcal cells to the action of the phage-associated lysin.     

Scanning Electron Microscopic Studies of Candida albicans

A scanning electron microscopic study of selected morphological stages of Candida albicans is presented. Stages represented are budding yeast cells, mycelial-like forms, chlamydospores, germ tube formation, and an unusual rough cell type.     

Electron Microscopic Studies on Opaque Colony Variants of Group A Streptococci

Opaque colony variants of two strains of group A streptococci have been compared with blue colonies of the same strains by electron microscopy. In opaque colonies, the cocci are joined into elongated chains by exaggerated intercellular septa that often occupy the major portion of each cell's circumference. The thickness and lamination of cell walls in opaque colony variants are identical to those aspects of cell walls in blue colony forms. The similarity in cell wall architecture is found between opaque and blue forms whether or not M protein (and M associated surface fimbriae) is present. Extensive, direct contact between the nucleoid and the cytoplasmic membrane beneath intercellular septa is seen in opaque colony variants. The relationship of this marked nucleoid-cytoplasmic membrane association to the unusual chain forms in the opaque colony variants is unclear.     

Scanning Electron Microscopic Studies on the Organ Development of

The pattern of development of the floral parts of Longan flower was followed using scanning electron microscope. Floral initiation begins with the formation of calyx protrusions around the floral apex. After the calyx protrusions have appeared, the petal primordia at the base of the floral apex start to appear and then followed by the androecium primordia which appear at the periphery of the floral apex. Gynoecium formation begins much later (at about 30 days after floral initiation). In the male flower, androecium develops normally forming anthers and filaments. Anthers also develop in the female flowers but they are smaller and the filaments much shorter. Gynoecium in the female flower is well developed and when mature it produces a long style, a two-prong-stigma and two ovaries. In the male flower the gynoecium is poorly developed the style is short and the stigma seldom splits. Ovaries are also poorly developed in the male flower. In addition to male and female flowers, Longan also forms a number of abnormal flowers with poorly developed androecium and gyn6ecium. Male and female flowers only become apparent at about 40 days after the initiation of flower differentiation. Prior to this it is difficult to know whether a particular developing flower is going ultimately to become a male or female flower. The formation of abnormal flowers also become obvious' at about 40 days after the initiation of flower differentation.     

A Disrupting Factor in Silver Staining Techniques

Single-stage surface replicas of treated or fresh pollen grains can be made ready for the electron microscope in 1.5 hr. The microspores are discharged into a drop of 50% acetone on a 1 cm square of cleaved mica and air dried. Carbon is evaporated to a film thickness of 35 mμ during rotation of the mica support. The carbon film and microspores are parted from the mica with water and heated in 2-aminoethanol at 145-155 C for 10 min to 3 hr. The replicas are then washed 5 min or longer on water at 90 C and picked up on electron microscope grids. The resulting self-shadowed surface replica can be immediately observed by electron microscopy.     

A Comparison Between Different Methods for the Determination of Reduced and Oxidized Glutathione in Mammalian Tissues

In this study, three rapid assay techniques for the determination of glutathione, one enzymatic, one flu-orometric and one newly patented colorimetric method, were compared by measuring reduced (GSH) and oxidized (GSSG) glutathione in guinea-pig heart and liver. The HPLC technique was used as a standard, since it is considered the most reliable assay method. In heart, all methods measured the same levels of GSH (about 1 µmole/g wet tissue), whereas in liver the fluorometric assay gave GSH levels about half as high as those measured by the other methods (about 3 vs. 7 µmoles/g wet tissue). Conversely, the fluorometric assay grossly overestimated GSSG concentration (by 5 to 8 times) in both heart and liver. These results confirm previous doubts about the use of the fluorometric technique for GSSC determination in mammalian tissues and also raise some questions about its use for the measurement of GSH in liver. In this tissue, the GSH concentration determined by the fluorometric method was shown to be inversely correlated with the size of the sample, suggesting the presence of some quenching material.     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

Electron Microscopic Studies on the Intra-flagellar Structures of Trypanosomes*

SYNOPSIS. The intraflagellar structure (IFS) of the flagella of Trypanosoma brucei was examined on the basis of ultrathin sections in various planes. The IFS is composed of filaments approximately 50 A thick. These filaments seem to be identical with the protofilaments found earlier to be the basic elements of the contractile flagellar fibrils. The fibrillar system is firmly connected with the IFS and the latter is attached to the flagellar membrane by filaments. The lattice-like appearance of the IFS is caused by longitudinal and oblique filaments running in different planes. The structure of this network is discussed in detail. The IFS may serve as an abutment for the contractile flagellar fibrils.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

Hematoxylin Staining of Tissues Embedded in Epoxy Resins

Sections of 0.5-2 μ thickness are affixed to slides with albumen adhesive, thoroughly dried, and placed in xylene or toluene for 1 hr, then brought through ethanol to water. Sections of tissue fixed in O_sO₄ are treated first in 0.1% KMnO₄, then with 1.0% oxalic acid, and after rinsing, incubated at 60 C for 12-24 hr in hematoxylin (Harris's or Ehrlich's) and counterstained 10-15 min with 0.5% phloxine B. Permanent preparations are made by clearing and mounting in a synthetic resin. The method requires only easily available reagents and is suitable for routine processing of epoxy sections.     

Electron Microscopic Studies on Intracellular Multiplication of Rickettsia tsutsugamushi in L Cells

The mechanism and kinetics of intracellular growth of Rickettsia tsutsugamushi were investigated by electron microscopic observations, parallel with quantitative analysis by counting the rickettsiae seen in electron micrographs and by plaque assay for infectivity of the culture. The observations demonstrated the existence of electron-less dense and -dense types of rickettsiae in the early stage of infection, binary fission and the process of release of the microorganisms in the host cell cytoplasm and from the cell surface, formation of abnormally long rickettsiae, and the process of lysis of the host cell in the later stage of infection with vacuole formation between the inner and outer leaflets of the host cell nuclear membrane. Separate titrations of infectivity of the cells and the culture fluid showed a very slow increase in infectivity in the culture fluid compared with the intracellular titer, suggesting that the progeny rickettsiae stay in the cell or at the cell surface for a relatively long period. Doubling time of the rickettsia was found to be about 9 hr.     

Electron Microscopic and Histochemical Studies of Sporozoite Formation in Plasmodium berghei*

SYNOPSIS. Observations were made on the differentiation of fine structure during sporogonic development of Plasmodium berghei. The oocyst in the process of sporozoite formation is an encapsulated structure 30-40 μ in diameter. It typically develops while in an extracellular position, attached to the basement membrane of the mosquito midgut and projecting into the mosquito hemocoel. Occasionally, however, ookinetes passing thru the midgut epithelial cells may become impacted within a cell so that the resulting oocyst develops intracellularly. Each oocyst has a large differentiating region, the sporoblastoid body. This body contains large dividing nuclei which are Feulgenpositive, and a cytoplasm which includes mitochondria, dense rodlike structures, cytoplasmic membranes, cisternae and vacuolar structures, Golgi material, and ribosomes which are both free and membrane-associated. Sporozoite budding takes place along the surface of the sporoblastoid body. Bits of a new membrane condense under the plasma membrane which bounds the sporoblastoid body. These 2-membraned sites then bulge out, continue to elongate, and eventually become sporozoites. The various nuclear and cytoplasmic components of the sporoblastoid body are passed into the sporozoites during their elongation. In addition, the sporozoite develops a system of elogate, subpellicular microtubules, possibly contractile in function. The pellicle of the sporozoite is broken by an opening, the cytostome (micropyle). The anterior end is truncate.     

A Mixture of Paraphenylenediamine and Imidazole: Its Effect on the Extraction of Lipid Droplets during Electron Microscopy Staining

To prevent extraction of lipids during a double staining procedure for electron microscopy, the tissue slices, double fixed with glutaraldehyde and osmium tetroxide to preserve microvesicular lipid droplets in the cytoplasm, were immersed for 2 hr in veronal buffer (pH 9.0) containing 0.5% p-phenylenediamine and 0.5% imidazole immediately after postfixation. The stained sections of the immersed tissue slice showed blackened, well circumscribed lipid droplets similar to those in corresponding unstained sections. Moreover, highly contrasting features of the cellular architecture could be visualized with the double stained, as well as routinely prepared sections.