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Glycol Methacrylate Embedding or Light Microscopy
Abstract
A technic for preparing tissue sections for plastic embedding and the underlying process of polymerization is described. This technic involves the use of glycol methacrylate with a catalyst, benzoyl peroxide, and an initiator, N,N-dimethylaniline. Material prepared using this method is exceptionally well preserved, and suitable for either histological or histochemical staining.
... Polymerisation of BMM (and LR White -see the TEM chapter by Chaffey in this volume) is an exothermic reaction (Lulham, 1979;Baskin et al., 1992), and, if too rapid, may result in the formation of gas bubbles in (Bennett et al., 1976;Lulham, 1979;Fig. 10b in Glauert and Young, 1989), and/or incomplete polymerisation of, the resin. ...
... Polymerisation of BMM (and LR White -see the TEM chapter by Chaffey in this volume) is an exothermic reaction (Lulham, 1979;Baskin et al., 1992), and, if too rapid, may result in the formation of gas bubbles in (Bennett et al., 1976;Lulham, 1979;Fig. 10b in Glauert and Young, 1989), and/or incomplete polymerisation of, the resin. ...
Aspects of the biology of the cytoskeleton of plant cells are briefly summarised, and techniques for its study introduced. Detailed protocols are provided for chemical-and freeze-fixation, processing, embedding, immunolocalisa-tion, and epifluorescent visualisation of microtubules and microfilaments in the secondary vascular system of angio-sperm trees embedded in either low melting point Steedman's wax or butyl-methylmethacrylate. Results of epifluorescence visualisation of the microtubule and microfilament cytoskeleton during wood formation in horse-chestnut (Aesculus hippocastanum L.) are summarised, and future directions of cytoskeletal studies on angiosperm trees considered.
... To ensure recovery of a wide variety of endogenous developmental stages the snakes was orally inoculated with oocysts three times, at nine days intervals and was sacrificed 61 days after the first ingestion, immediately following recovery of oocysts in the faeces. Samples from the gut tissue were fixed in 10 % neutral buffered formalin and embedded in glycol methacrylate (Lulham, 1979). Sections, 3-4 µm thick, were cut with a glass knife on a Sorvai JB4 microtome and were stained with Meyer's haemalum, using eosin or eosin and phloxin as counterstains. ...
Des oocystes de taille et de forme identiques a ceux de Caryospora colubris Matushka, 1984 sont excretes par le serpent Coluber jugularis L. d'Israel. Des tentatives d'infection de Souris blanches avec des oocystes ont echoue. Le cycle de developpement endogene du parasite de C. colubris est decrit chez un serpent infecte experimentalement per os avec des oocytes provenant d'une infection naturelle.
... Infected geckoes, 14 Hemidactylus turcicus L. 1766, out of 28 collected from urban and rural locations throughout Israel, two Hemidactylus mabuia (Moreau de Jonnes 1818) and one Pachydactylus capensis Smith 1846, captured from suburban dwellings near Pretoria, South Africa, were necropsied for the study of endogenous stages. Samples from the intestine, liver and gall bladder of the necropsied geckoes were fixed for histology in 10% buffered neutral formalin and embedded in glycolmethacrylate (Lulham 1979). Sections 3--4 f.Lm thick, cut with a JB4 Sorvall glass knife microtome were stained with either Meyer's haemalum and counterstained with phloxine and eosin (vide H-E) or in Schiffs Periodic Acid method (vide P.A.S.). ...
new genera are proposed to accomodate new and previously described species of eimerian coccidia from reptiles which undergo endogenous development either in the bile epithelium — Choloœimoria n. gen., or in the microvillous zone of the intestinal epithelium — Acroeimeria n. gen. Endogenous development is described from 3 species, all from geckoes: C. turcicus (syn. Eimeria turcicus Upton, McAllister and Freed, 1988) from Hemidactylus turcicus in Israel; C. pachydactyli n. sp. from Pachydactylus capensis in South Africa and A. lineri (syn. Eimeria linen McAllister, Upton and Freed, 1988) from H. turcicus, Israel and H. mabouia, South Africa. Biliary epithelial cells infected by Choleœimeria become hypertrophic and are displaced to the surface of the epithelial layer. Oocysts are cyllndroid to oval, lack a stieda body and sporulate in the gall bladder. The developing endogenous stages of Acroeimeria, enclosed in the microvillous border of the host cell, expand into the intestinal lumen. Oocysts are oval-spherical, lack a stieda body and sporulation is exogenous.Twee nuwe genera word voorgestel vir nuwe sowel as reeds beskryfde spesies van reptiel Coccidia wat endogene ontwikkeling in òf die milt — Choleoeimeria n. gen., òf in die mikrovilli-sone van die intestinale epiteel — Acroeimeria n. gen. ondergaan. Endogene ontwikkeling word vir drie spesies beskryf wat almal in geitjies voorkom, d.i. C. turcicus (sin. Eimeria turcicus Upton, McAllister en Freed, 1988) van Hemidactylus turcicus in Israel; C. pachydactyli n. sp. van Pachydactylus capensis in Suid-Afrika en A. lineri (sin. Eimeria lineri McAllister, Upton en Freed, 1988) van H. turcicus, Israel en H. mabouia, Suid-Afrika. Galepiteelselle, besmet met Choleoeimeria, word hipertrofies en na die oppervlak van die epiteellaag verplaas. Oösiste is silindries tot ovaal, die stieda-liggaam is afwesig en sporulasie kom in die galblaas voor. Die ontwikkelende endogene stadium van Acroeimeria, ingesluit in die rand van die mikrovilli van die gasheerselle, verleng tot in die intestinale lumen. Die oösiste is ovaal-sferies, ’n stieda-liggaam is afwesig en sporulasie is eksogeen.
... In both Placobdella multistrigata and P, costata, the gastric ceaca contents included released trophozoites MATERIALS AND METHODS and encapsulated, rarely released gametocytes, but never further developmental stages. In both leeches, Leeches fixed in neutral buffered formalin (10%) macrogametocytes associated with microgametocytes were processed for methyl-methacrylate/glass knife and zygotes were found attached to the surface of the histology by the Lulham (1979) method. Sections, 3 to 4 epithelium of the intestine (Figs. 1 to 6). ...
... Intestinal smears were air dried, fixed in methanol and stained in Giemsa (1 : 4, pH 6.8) for 20 minutes. Histological preparations made from intestines and whole lish fry were fixed in cold 10 % (v/v) buffered neutral formalin and embedded in glycol methacrylate (Lulham, 1979). 3-6 μm sections were cut by a Sorval JB-4 glass knife microtome and were stained either by Mayer's haemalum (counterstained with eosin alone or initially with phloxine) or by Giemsa and PAS methods adapted to methacrylate sections (Du Pont, 1981). ...
Eimeria (s. l.) vanasi n. sp. is described from the intestine of a variety of cichlids in Israel and South Africa. Merogony and gamogony stages are both intra- and epiepithelial, Sporogony is exogenous with young zygotes being released from the fish in the faeces. Fully developed sporocysts are ellipsoid and apparently lacking both a Stieda body (characteristic of Eimeria s. st. and Epieimeria species) and a suture line (characteristic of Goussia species). In view of the present controversial generic status of piscine coccidia, the species is tentatively designated as Eimeria (s. l.) vanasi n. sp. (Eimeriidae, Apicomplexa, Protozoa).
Recently, research efforts are increasing toward the development of elastomeric materials with superior mechanical properties and biocompatibility in the degradation profile. In this chapter, the overview of biodegradability tests along with preparation strategies of the elastomers is outlined. The potential application in the medical field; soft tissue engineering and drug delivery require the compatibility in the mechanical properties of the polymer scaffold and the tissues to be grown. This chapter provides an update on biodegradable elastomeric materials in terms of their relevance in biomedical application.
. A lymphocystis infection is described from aquarium-held snakeskin gourami, Trichogaster pectoralis (Regan). The cytoplasm of the hypertrophic cells revealed virus particles of extended hexagonal profiles with thin two-layered capsids. Subsequent examination of the fish 4 months later revealed hypertrophic cells showing varying degrees of collapse and degradation. The virus particles loading such cells were of regular hexagonal profiles with thick three-layered capsids.
Patent infections with Schellackia cf. agamae were established in the starred lizard Agama stellio by feeding blood or liver or by injection of blood from A. stellio infected with sporozoite stages. Mosquitoes, Aedes aegypti and Culex molestus, and larval ticks of Hyalomma aegyptium were fed on sporozoite infected A. stellio and themselves gave rise to patent infections after ingestion by uninfected A. stellio. Merogony in the duodenal epithelium usually lasted to the 14th day post-infection (d.p.i.), while in some lizards it persisted until 18 d.p.i. Microgamonts, macrogamonts and zygotes occurred 11–14 d.p.i. Oocysts migrated into the lamina propria before sporulation. Sporulation had terminated by 18 d.p.i., when sporozoites first appeared in the liver and the blood. Small, young sporozoites invaded hepatocytes and liver macrophages, while a few occurred in the erythrocytes. Sporozoites which occurred from 25 d.p.i. were larger and had invaded erythrocytes and aggregated in melanin-containing liver macrophages. Parasitaemias of medium and high levels as well as hepatic sporozoites persisted in some experimentally infected lizards for 357–370 d.p.i. Lizards could not be reinfected while a parasitaemia was detectable in them. Reptiles other than A. stellio could not be infected with S. cf. agamae.
Histopathological changes involved in Ichthyophthirius multifiliis infections are described from a wide range of host fishes—carp, crucian carp hydrids, Carassius carassius×C. auralus, goldfish, eels, African catfish, Glorias lazera, several species of cichlid fishes and rainbow trout. Fish were obtained from diverse geographical regions: Israel, South Africa, Portugal and Venezuela, as well as from laboratory experimental infections. Observed histopathological changes could not demonstrate any interspecific differences, but individual fish or different groups of fish of the same species demonstrated wide variability in susceptibility to infection and consequently in the nature and severity of the histopathological response. Low to moderate primary infection and secondary reinfestations did not induce significant histopathological changes in the integument. The proliferative response of the integumentary epithelium appeared only following subsequent infections. Repeated heavy reinfections induced massive cellular necrosis and in very heavy infections extensive lysis of the epithelial tissue. Host-parasite interactions in the light of the histological findings are discussed.
Red drum Sciaenops ocellatus is a new fish species farmed in Mediterranean and Red Sea mariculture facilities in Israel. The success of this introduced species in Israel will undoubtedly encourage expansion of red drum farming also to other Mediterranean countries. In the present study, red drum was shown to be susceptible to myxidiosis, a serious disease of cultured sparids. The responsible agent, Myxidium leei, has been causing increasing economical losses to cultured sea bream Sparus aurata throughout the Mediterranean basin. Red drum of mean weight 69.7 g (range 23–117 g) were cohabited with infected sea bream or exposed to water effluent discharged from the same fish. Forty three days after initial exposure, trophozoites, sporoblasts and spores identified as M. leei were found developing in the posterior gut of 45.8% and 35.0% of the test fish in the respective treatments. The early stages of infection and parasite development in the gut mucosa epithelium of red drum were similar to those previously observed in sea bream. M. leei has a low degree of host specificity and should be regarded a potential threat not only to members of the Sparidae, but also to other warmwater cultured fish species in the Mediterranean. Although fish stocks from Mediterranean waters have been introduced into the Caribbean, M. leei has hitherto not been reported from North America. It is advisable to exercise extreme caution with future Mediterranean introductions so as to prevent accidental transemination of this myxosporean into the US Gulf Coast states, in which red drum is a commonly farmed species, as there is presently no available treatment for myxidiosis.
Eimeria gastrosauris n. sp. exhibited endogenous development in the stomach lining and secretory epithelia of Heteronotia binoei from the Mt Isa region, northern Queensland. Morphologically similar coccidians were found in the stomach of Oedura monilis from the Mt Speke region, northern Queensland. Ocysts were oblong-ellipsoids, with bivalved sporocysts, two distinct residua and endogenous sporulation. Ocysts of similar shape and size were also found in faeces of Gehyra australis from the Townsville region of northern Queensland. Developmental stages, consisting of meronts, merozoites, microgamonts, macrogamonts and developing ocysts, were observed in H. binoei and O. monilis, and are described.
Goussia cichlidarum n. sp. (Barrouxiidae, Apicomplexa) is a coccidian parasite found in the epithelial lining of the swimbladder in a variety of cichlid fish, most commonly inSarotherodon galilaeus. Infected fish were found in Lake Kinneret and in fish ponds in Israel and in Uganda. Natural infections were investigated as well as experimental infections obtained in the laboratory. Merogony and gamogony is completed within a hypertrophic host cell. The latter is displaced to the surface of the epithelial layer and during the parasitization process is reduced to a membranous envelope. This cell remains attached to the epithelial layer up until the stage of oogony. Oocysts complete their differentiation free in the swimbladder lumen.
Two new species of Isospora are described from skinks, I. cryptoblephari n. sp. in Cryptoblepharus virgatus and I. delmae n. sp. in Delma nasuta, both collected in Australia. I. cryptoblephari ocysts are ellipsoidal to subspherical, 17.5–22.5 25.0–30.0 m with two ovoid sporocysts, 9.0–10.0 12.5–14.0 m. I. delmae ocysts are spherical to subspherical, 16.5–19.0 16.5–20.0 m with two ovoid sporocysts, 5.0–6.5 9.0–12.5 m. These species of Isospora had two sporocysts, each containing four sporozoites and a characteristic Stieda body. A study of endogenous stages in the host's intestine revealed that I. cryptoblephari develops in the nucleus and I. delmae in the cytoplasm of the host's gut epithelial cell. In the former, both merogony and gamogony occurred in the nucleus.
Three new species of Isospora Schneider, 1881 are described from agamid lizards, Isospora cannoni n. sp. in Diporiphora australis from northern Queensland, Australia, I. choochotei n. sp. in Calotes mystaceus from northern Thailand, and I. deserti n. sp. in Agama pallida from Israel. I. cannoni ocysts are subspherical, 20–25 22.5–27.5 m with two ovoid sporocysts, 14–15.5 10–11.5 m. I. choochotei ocysts are spherical to subspherical, 24–32 28–32.5 m with two ovoid sporocysts, 11 15.5–18 m. I. deserti ocysts are spherical, 25–28 m in diameter with two ovoid sporocysts, 10–11 14–17.5 m. All species had sporocysts with Stieda bodies and underwent endogenous development in the nucleus of the host gut epithelial cells. At completion of merogony and gamogony, the host nucleus was reduced to a thin envelope. The significance of endogenous stage characteristics in Isospora taxonomy is discussed.
Lungs from 48 feedlot cattle that had died from bacterial pneumonia were examined grossly and microscopically. Criteria based on microscopic lesions were adopted to age these pneumonias. In 38 cases, pneumonic lesions were of relatively uniform age throughout the affected tissue. In eight other cases, the presence of older lesions confined to one or two lobes suggested a previous episode of pneumonia. The aging criteria adopted were in agreement with the duration of the observed clinical signs in 26 cases. In 13 other cases, the pneumonia was estimated to be of longer duration than suggested by the history, whereas in the remaining nine cases, it was estimated to be more recent. Areas of tan discoloration of the parenchyma surrounded by white or yellow borders were considered the best areas to examine microscopically since they offered the best chances of revealing necrosis and fibrosis, the main lesions used to age the pneumonia.
A new microsporidian infecting the Mediterranean common stingray Dasyatis pastinaca (Linnaeus, 1758) is described from Iskenderun Bay, Turkey. The parasite invades the disc muscles, producing slender, spindle-shaped subcutaneous swellings that develop into massive, elongated, tumor-like protuberances measuring up to 11 x 4 cm. Severity of the infection may vary from light (1 or 2 small lesions) to intense, with large parts of the dorsal surface covered with lumps and protrusions. These masses contained a yellowish-white caseous substance consisting of degraded host tissue and microsporidian sporophorous vesicles, which in turn contained developing sporonts, sporoblasts and spores. The ripe spore contained a uni-nucleate sporoplasm and large posterior vacuole, and measured 3.8-4.3 x 2.6-2.8 microm. Infection prevalence was 21% in a sample of 143 host individuals examined. All the infected stingray individuals were within the weight class of 300 to 800 g (200 to 305 mm disc width). Phylogenetic analyses of rDNA sequences indicate that this microsporidian belongs to the Pleistophoridae and clusters with species of the genera Ovipleistophora Pekkarinen, Lom & Nilsen, 2002 and Heterosporis Schubert, 1969. However, the morphology, development and host differ distinctly from all reported species, including those belonging to these 2 genera, and it is thus assigned to a newly erected genus and named Dasyatispora levantinae gen. et sp. nov. This is the first record of a microsporidian infection in a batoid. It is also the first microsporidian species to be formally described from an elasmobranch.
A method for selective staining of flavan-3-ols in plant tissues fixed with glutaraldehyde is given. The use of glycolmethacrylate as embedding medium allows the sulphuric acid-containing staining solution to be heated without destroying the fine structure of the tissue. The distribution of flavan-3-ols and proanthocyanidins in different plant tissues is discussed.
A method has been developed for the histologic evaluation of rat inner ear using glycol methacrylate (GMA) and steel knife sectioning. The necropsy, fixation, and histologic techniques described are not so complex and difficult as to preclude their routine use. The method is particularly useful in studies in which the entire inner ear of a large number of animals must be evaluated histologically.
To evaluate the use of methyl methacrylate resin as an embedding medium for undecalcified bone marrow trephine biopsy specimens.
About 2500 undecalcified bone marrow trephine biopsy specimens were processed, and embedded in methyl methacrylate resin. Semithin sections (2-3 microns) were stained by routine tinctorial and immunocytochemical staining methods with a wide range of antibodies using a standard streptavidin biotin horseradish peroxidase technique. Different antigen retrieval pretreatments were evaluated.
Bone marrow trephine biopsy specimens are embedded routinely in methyl methacrylate at the Haematological Malignancy Diagnostic Service at The Leeds General Infirmary. Over 50 different primary antibodies are in current use; for the majority of these, microwave antigen retrieval or trypsin digestion, or both, is either essential or greatly enhances the results.
Embedding bone marrow trephine biopsy specimens in methyl methacrylate resin retains morphology and permits reliable, high quality immunocytochemistry. This is particularly desirable for the demonstration of neoplastic cells in regenerative marrow after chemotherapy, and in the detection of residual disease after treatment. The use of methyl methacrylate for routine use on bone marrow trephine biopsy specimens is advocated.
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