Giardia muris: scanning electron microscopy of in vitro excystation

A recently developed in vitro excystation procedure results in almost total excystation of Giardia muris, an intestinal parasite of mice. The present experiment examines the G. muris cyst morphology by scanning electron microscopy and the efficacy of the excystation procedure. Untreated cysts of G. muris were elliptical and displayed a distinctive surface structure. Excystation began almost immediately after incubation had begun and most trophozoites emerged within 30 min. Excystation appears to involve flagellar action of the encysted trophozoite. A tear of the wall occurred at one pole. This opening was subsequently enlarged, presumably by flagellar action. Trophozoites emerged, posterior end first, and an associated mucoid-like material was extruded. Newly emerged trophozoites were nearly oval in shape. Trophozoites quickly became flattened, elongate, and underwent cytokinesis resulting in two daughter trophozoites. Few organisms not excysted were seen after 30 min incubation.     

Morphology of the cyst of Giardia microti by light and electron microscopy

Cysts of Giardia microti, isolated from feces and intestinal contents of Microtus ochrogaster, were examined by light and electron microscopy. These cysts differed morphologically from cysts of other G. duodenalis morphological types in that these cysts often contained two apparently differentiated trophozoites with mature ventral discs. Cysts more closely resembling those reported for G. lamblia and G. muris were in greater abundance in preparations made from intestinal contents and were interpreted as immature cysts. "Multiple fission" cysts, reported in G. muris and G. microti by earlier workers, were not observed; however, endosymbiotic bacteria were found in the cysts of G. microti and may have been responsible for reports of multiple fission in the cysts of Giardia.     

Morphology of Giardia agilis: Observation by Scanning Electron Microscopy and Interference Reflexion Microscopy1

The flagellated protozoan, Giardia agilis, was isolated from tadpole small intestine and examined by scanning electron microscopy and interference reflexion microscopy. The general morphology of the G. agilis trophozoite is similar to G. muris and G. duodenalis, but with modifications that reflect its elongated form. Interference reflexion microscopic analysis of attachment of G. agilis reveals a pattern of focal contacts by the lateral crest of the ventral disc, the ventrolateral flange, the lateral shield, and by numerous microvillus-like appendages found along the lateral border of the trophozoite. The pattern of focal contacts was observed to be dynamic; trophozoites were observed to make and break the focal contacts in a relatively short time and to glide along the surface of the substratum without breaking focal contacts.     

Topographic examination of sister chromatid differential staining by nomarski interference microscopy and scanning electron microscopy

BrdU-substituted Chinese hamster chromosomes were treated with a hot Na₂HPO₄ solution and stained with Giemsa to produce sister chromatid differential staining (SCD). The process of SCD was examined with the Nomarski differential interference microscope and the scanning electron microscope. After the Na₂HPO₄ treatment alone, unifilarly BrdU-substituted (TB) chromatids appeared somewhat more severely collapsed than the bifilarly substituted (BB) chromatids. Subsequent Giemsa staining, however, brought about pronounced piling up of the Giemsa dye on the TB-chromatids but not on the BB-ones, causing highly distinct differential Giemsa staining as well as a marked differentiation in surface topography between the sister chromatids. Removal of the Giemsa dye from the differentially Giemsa stained chromosomes resulted in a disappearance of such a pronounced topographic differentiation.     

In vitro excystation of Giardia from humans: a scanning electron microscopy study

The in vitro excystation of Giardia lamblia on cysts isolated from human feces was studied. After purification by sucrose gradient, cysts were incubated in a pepsin-acid solution, then placed in a modified HSP3 medium where excystation occurred within a few minutes. The excystation procedure was studied by continuous observations by light microscopy and sequential observations by scanning electron microscopy (SEM). The in vitro excystation was stopped at timed intervals during incubation by addition of a large amount of 1% glutaraldehyde. The excystation process began by the cyst wall opening at one pole. Flagella protruded rapidly, the parasite emerged progressively from the cyst envelope, posterior end first, the empty cyst collapsed and shrank. Although flagella emerging from the organism were distinguishable, the cell body had not yet shown all the morphological features of the G. lamblia trophozoite. A radical rearrangement of the organism occurred gradually: initially oval in shape, the parasite became round, then elongated, flattened, and underwent cytokinesis. The daughter trophozoites acquired their typical morphological features: the shape, the adhesive disc with the C-shaped structure distinctly visible on the ventral surface, and the definite placement of the flagella. These observations obtained on G. lamblia by SEM were comparable to those obtained with G. muris.     

Morphology of the second- and third-instar larvae of Dermatobia hominis by scanning electron microscopy

Larvae of Dermatobia hominis 10–27 days old were collected from experimentally infected rats and their morphology was studied by scanning electron microscopy. The moult from the second to third instar occurs at 18 days, with emergence from the host at 30 days post-infection. The second-instar larvae bear on the pseudocephalon, antennae (coeloconic sensilla), and coeloconic and basicoconic sensilla on the maxillary sensory complex. The thoracic segments bear small backwardly-directed spines anteriorly and ventral trichoid and campaniform sensilla. The first four abdominal segments have small and large backwardly-directed spines that are absent on segments five and six. The seventh and eighth abdominal segments have medium-sized forwardly-directed spines. Abdominal segments are encircled by campaniform sensilla. The terminal end of the eighth abdominal segment bears the anus, prominent anal lobes and two spiracular openings on each spiracular plate. Spiracular plates show a radial sun ray pattern. The rear abdomen also bears an ecdysal aperture, several pores and eight coeloconic sensilla. Although there are slight morphological differences, the spines (predominantly flat and thorn-like) and sensilla (campaniform and coeloconic) of the third-instar larvae show a similar arrangement to that of second-instar larvae. Thoracic trichoid sensilla are not seen in third-instar larvae. A perispiracular gland aperture is situated above each posterior spiracular opening. These morphological features are compared with those of other cuterebrid larvae.     

Nucleopolyhedrovirus: scanning electron microscopy technique

A simplified methodology was developed to study the geometric form of multiple Bombyx mori Nucleopolyhedrovirus by scanning electron microscopy. The virus belongs to Baculoviridae family and was isolated from the silkworm Bombyx mori (L.) (Lepidoptera: Bombycidae). The polyhedra of Nucleopolyhedrovirus were obtained from the filtrate, inoculum and hemolymph of the silkworm experimentally infected with nuclear polyhedra. This material was placed on stubs, where a copper tape was previously adhered. After dry at room temperature the virus was covered with carbon and gold. Scanning electron microscopy analysis revealed a well defined morphology for the polyhedra of multiple Bombyx mori Nucleopolyhedrovirus, making possible the mathematical study that identified it as a truncated octahedron. The form of the polyhedron can present taxonomic value, once it is specific for each viral lineage.     

Fish chromosomes: a display by scanning electron microscopy

This note reports the first display of fish chromosomes by scanning electron microscopy and assesses the value of this technique to fish karyology.     

Transmission electron microscopy of tissue prepared for scanning electron microscopy by ethanol-cryofracturing.

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of specimens cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

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.     

Optical and scanning electron microscopy observation of the mucosa of human fetal tongue

The structural features of the human foetal tongue have been studied in foetuses from 8th to 20th week of pregnancy. The characteristics of the developing papillae as well as of epithelial and mesenchymal layers have been pointed out. An early differentiation of the mesenchymal tissue has been observed, concerning phenomena of cellular condensation and reticular fibers organization both in superficial and deep layers. The hypothesis of the existence of straight interactions between epithelium and mesenchyme also in the developing human tongue mucosa has been suggested. Also the observations at SEM demonstrate that from the 8th to the 20th week the epithelial surface of the tongue reaches a stable structural pattern. From 11th week a characteristic cellular polymorphism occurs: cells with microvilli that diminish progressively, ciliated cells that disappear almost completely at the 20th week and cells whose free surface show microplicae, definitive stage of the tongue cell evolution.     

Mitochondrial structure revealed by high-resolution scanning electron microscopy

Mitochondrial structure has been examined in three dimensions using high-resolution scanning electron microscopy in cells from rat liver, retina (photoreceptors and retinal pigment epithelium), and kidney (proximal convoluted tubular cells and podocytes). Tissues were prepared by aldehyde-osmium fixation and freeze cleavage using a cryoprotectant, followed by removal of the cytosol by immersion in a dilute osmium tetroxide solution. The microscope used (Hitachi S-570) was equipped with a secondary electron detector located in the column above the specimen, situated within the objective lens. Mitochondria in all tissues examined were found to have only tubular cristae, which in some instances could be seen to span the entire diameter of the organelle. The walls of the tubular cristae, when unfractured, were in contact with the inner mitochondrial membrane; and their lumens were open to the intermembranous space. We hypothesize that in cells of many, perhaps most tissues, mitochondrial cristae are not shelf-like but are, in fact, tubes which span the mitochondrial matrix and are continuous with the inner mitochondrial membrane at both ends.