Osmium Postfixation Research Articles

The Differences in Lung Ultrastructural Response of Small and Large Rats to Hyperoxia

In the work with oxygen exposed animals, it is readily apparent that there are great differences in the responses of young and old animals. Young rats are far more resistant to oxygen poisoning than are older rats. The younger animals, weighing 90-110 grams (about four weeks old) will live in 100% oxygen at one atmosphere for long periods of time, many surviving for four to six weeks. On the other hand, larger rats, 300-350 grams (12-16 weeks old) live only about three days in the same atmosphere. Obviously, changes must occur at a greater rate, they may be more damaging and may even be different in character.Rats in the 90-110 gram range and in the 300-350 gram range were exposed for 48 hours to pure oxygen at one atmosphere. The animals were sacrificed, the tissue removed, cut into small blocks and fixed in glutaraldehyde, followed by osmium post-fixation.

Fine Structure and Organization of the Musculature in the Miracidium of Fasciola hepatica

The muscle fibers of the miracidium lie just beneath the surface layers of the body and apical papilla. The muscles of the body comprise outer circular and inner longitudinal fibers. The muscles of the apical papilla are similarly arranged but there are only six longitudinal fibers which serve as papillar retractors. Each of the six fibers is inserted at the base of one of the six lateral papillae. The fine structure of the fibers is similar to that of other nonstriated platyhelminth fibers having thick and thin myofilaments. Dense bodies are not, however, present and insertion of thin myofilaments has not been determined. The fibers appear to be anucleate but have well-developed sarcoplasmic reticulum, and mitochondria and glycogen are present in the sarcoplasm. Desmosomes occur between the circular and longitudinal fibers and between circular fibers and overlying cells of the body surface, particularly in the apical papilla but also in the body musculature. There have been several accounts of the fine structure of platyhelminth muscle. Freeliving planaria have been investigated by MacRae (1963, 1965) and Morita (1965); cestodes by Lumsden and Byram (1967); and digenetic trematode cercariae by Cardell and Philpott (1960), and Lumsden and Foor (1968). There is a basic similarity of structure but some variations exist particularly with regard to the presence and distribution of dense bodies in the fiber matrix. The fine structure of the body wall of the miracidium of Fasciola hepatica was described in an earlier paper (Wilson, 1969) and this work has been extended to a detailed investigation of the musculature of the miracidium. Collin (1968) has made a similar study of the muscle systems of hatched oncospheres of Hymenolepis citelli. Miracidia are plastic organisms and it has been assumed (Mattes, 1949; Dawes, 1960) that observed movements and changes of shape of the miracidial body during penetration of the snail host result from contractions of its musculature. A knowledge of the organization of the musculature is therefore a prerequisite for understanding the process of host penetration. MATERIALS AND METHODS Infected livers were obtained from the local slaughterhouse and mature flukes dissected out. Undeveloped eggs were teased from flukes into distilled water aand incubated for 12 days at 25 C in the dark. Mass hatching of miracidia into a Received for publication 18 March 1969. minimal volume of water was brought about by exposure to light. Miracidia were fixed at 4 C, 20 min after exposure of eggs to light, in either Zetterquist's buffered osmium tetroxide for 1 to 4 hr (after Kay, 1965), or 3% glutaraldehyde in cacodylate buffer pH 7.2 for 30 min, followed by postfixation in osmium for 4 hr. Miracidia were dehydrated in alcohol and embedded in an araldite-epon mixture. Prior to sectioning, individual miracidia were isolated in small pieces of cured resin and oriented transversely or longitudinally on top of a perspex rod, being fixed in position with araldite adhesive. Sections were cut on a Huxley ultramicrotome and collected on grids coated with formvar and carbon. Specimens were stained either with uranyl acetate during dehydration followed by lead citrate on the grid, or by both uranyl acetate and lead citrate on the grid. Sections were viewed in an AEI EM6B electron microscope operating at 60 kv.

Fine Structure of the Tegument of the Miracidium of Fasciola hepatica L.

The fine structure of the tegument of the miracidium of Fasciola hepatica is described. Ciliated epithelial cells overlie a thin discontinuous layer of cytoplasm. This is thickened between adjacent epithelial cells to form prominent intercellular ridges. The thin layer of cytoplasm is associated with diffuse extracellular fibrous material. Beneath the thin layer are circular and longitudinal muscle fibers and beneath these again are large (ca. 10, in diameter) vesiculated cells. The vesiculated cells communicate with the thin cytoplasmic layer by means of narrow cytoplasmic connections running between adjacent muscle fibers. Small electron-opaque vesicles are found in the vesiculated cells, thin cytoplasmic layer, and intercellular ridges. Flattened membranous bodies are also present in the intercellular ridges. The surface of the miracidium is covered with a layer of alcian blue-positive material which appears to be continuously secreted and is presumed to be acid mucopolysaccharide. The apical papilla has an array of circular and longitudinal muscle fibers attached to each other by numerous small desmosomes. The surface of the papilla lacks ciliated epithelial cells and is covered instead by a layer of corrugated cytoplasm. The structure of the miracidium of Fasciola hepatica has been described in detail at the light microscope level by Mattes (1949) and Dawes (1960), and there have been many studies on other miracidia (e.g. Price, 1931; Lynch, 1933; D6nges, 1964). The ultrastructure of the flame cells of the miracidium of Fasciola was described by Kiimmel (1958, 1959), and that of the miracidial eyespots by Kiimmel (1960), Isseroff (1964), and Isseroff and Cable (1968), but other aspects of fine structure do not appear to have been investigated. The above accounts give some indication of variations in structure, at least between miracidia from different families. Lee (1966) reviewed the structure and composition of the helminth body wall and pointed out that little work has been done on the fine structure of the body wall of larval trematodes. Descriptions of rediae have been given by Rees (1966), and Krupa et al. (1967); of a cercaria by Belton and Harris (1967); and of a daughter sporocyst by James et al. (1966). With the exception of the daughter sporocyst, the body surface was an anucleate cytoplasmic syncytium separated from the body wall musculature by an acellular basement membrane. In the case of the daughter sporocyst, the outer layer was nucleated. The miracidium stage in the digene life cycle has a body surface covered with discrete epithelial cells and Dawes (1960) has shown that, in Fasciola at Received for publication 17 June 1968. least, these cells are shed prior to penetration of the miracidium into the snail. This paper presents the results of an investigation into the fine structure of the tegument of the miracidium of Fasciola hepatica. MATERIALS AND METHODS Infected sheep livers were obtained from the local slaughterhouse and mature flukes dissected out. Undeveloped eggs were teased from flukes into distilled water and incubated for 12 days at 25 C in the dark. Mass hatching of miracidia into a minimal volume of water was brought about by exposure to light. Miracidia were fixed at 4 C, 20 min after exposure of eggs to light, in either Zetterquist's buffered osmium tetroxide for 1 to 4 hr (after Kay, 1965), or 3% glutaraldehyde in cacodylate buffer pH 7.2 for 30 min followed by postfixation in osmium for 4 hr. Miracidia were dehydrated in alcohol and embedded in an araldite-epon mixture. Prior to sectioning, individual miracidia were isolated in small pieces of cured resin and oriented transversely or longitudinally on top of a perspex rod, being fixed in position with araldite adhesive. Sections were cut on a Huxley ultramicrotome and collected on grids coated with formvar and carbon. Specimens were stained either with uranyl acetate during dehydration followed by lead citrate on the grid, or by both uranyl acetate and lead citrate on the grid. Sections were viewed in an AEI EMB6 electron microscope operating at 60 KV.

The role of buffers in lung fixation with glutaraldehyde and osmium tetroxide

In double-fixation of lung with glutaraldehyde and osmium, spherical electron dense granules appear as artifacts on the alveolar surface and on erythrocytes. We have tested the effect of six different buffers during glutaraldehyde prefixation, washing, and osmium postfixation. The granules were found to appear only when tissue prefixed in glutaraldehyde is postfixed with osmium in phosphate buffer. The possible significance of these granules is discussed. Further, the influence of different fixatives and stains on extracellular alveolar coatings as well as their possible relationship to pulmonary surfactant are discussed.

Periodate Treatment and “All-or-None” Dehydrogenase Staining Phenomena

It has been shown that when suspensions of fresh rodent sperm are incubated in the presence of tetranitro-blue tetrazolium (TNBT) and stained for succinic dehydrogenase (SDH),this enzyme is localized in the mitochondria of the middle-piece. Rat and mouse spermatozoa treated in this manner and postfixed (suspension) in 4°C Millonig's osmium tetroxide for brief periods demonstrate SDH reactivity in mitochondrial profiles that indicate no SDH, almost complete activity, or most commonly, intermediate staining reactions in the internal membranes (Figure 1). In the last instance, the cristal membranes display a heterogeneous formazan (reduced TNBT) staining pattern not in agreement with the Seligman “all-or-none” hypothesis concerning mitochondrial oxidative enzymatic reactivity.In order to test this hypothesis and circumvent some of the attendant difficulties in these procedures (penetration of reaction mixture components, fixatives), all cytochemical reactions and postfixation were performed on suspensions of sperm from the cauda epididymides of mature rats. An attempt was made to divorce electron densities due to osmium postfixation by employing 1% aqueous sodium periodate (pH 3-4) as a mitochondrial membrane stabilizer through the embedding procedures. Previous use of this treatment allowed preservation of the membranes of these organelles in positive contrast as evidenced by uranyl acetate staining of sectioned material (Figures 2 & 3).