Formalin-acetic Acid-alcohol Research Articles

Detection of the t(14;18) Translocation in Frozen and Formalin-fixed Tissue

As part of a retrospective study into the prevalence of the t(14;18) translocation in B-cell lymphomas, we assessed the suitability of the polymerase chain reaction (PCR) to amplify the t(14;18) major breakpoint region (MBR) in frozen and formalin-fixed tissue. Considering Southern blotting as a standard, the sensitivity of PCR was 81%. Of the various procedures used to extract DNA from paraffin-embedded tissue (PET), proteinase K digestion in the presence of nonionic detergents gave the highest yield and quality of DNA and the most efficient amplification rate. Using this method, excellent amplification rates (100%) were obtained for both the beta-globin control sequence and the MBR t(14;18) for fixed follicular lymphoma specimens collected in the previous 2 to 6 years (n = 27). Of nine older PETs, PCR on six gave inconsistent results, probably because of the poorer-quality substrate used for amplification. Specimens exposed to formol sublimate or formalin-acetic acid-alcohol were as suitable for amplification as tissues fixed in neutral-buffered formalin. The overall incidence of the MBR t(14;18) in all follicular lymphoma specimens as detected by both Southern blotting and PCR was 59% (23 of 39).

Deleterious effects of formalin/acetic acid/alcohol (FAA) fixation on the detection of HPV DNA by in situ hybridization and the polymerase chain reaction

As part of a study of archival cervical cancer specimens (1920s to 1980s) to determine whether changes have occurred in the prevalence of human papillomavirus (HPV) DNAs, investigations were performed on tissues which had been fixed in either 10% buffered formalin (NBF) or formalin-acetic acid-alcohol (FAA). HPV DNA was detected by in situ hybridization (ISH) using HPV 6, 11, 16 and 18 32P-labelled DNA probes under conditions of high stringency; and by the polymerase chain reaction (PCR) using 20-mer oligonucleotide primers to amplify 109 bases of the E6 region of HPV 16. In some instances results obtained from Southern blot hybridizations, which had been carried out on specimens of fresh cancer tissue, were available for comparison. When tissues had been fixed in NBF, HPV DNA sequences were detected in 53% of specimens by ISH and in 72% by PCR. In comparison, the rates of detection of HPV by ISH and PCR in tissues fixed in FAA were 17% and 21% respectively. These results indicate that FAA is clearly inferior to NBF for the preservation of detectable HPV DNA sequences in tissue sections.

The time course of the induction of callose in wounded pea roots

Initial study indicated that much callose is produced in pea root tissues during the preparation of fresh hand-cut sections or during conventional fixation in formalin-acetic acid-alcohol, glutaraldehyde or acrolein. In contrast, there is little callose in freeze-substituted tissues and this is mostly in sieve tubes and considered endogenous. Freeze-substitution was subsequently used to monitor wound-induced callose development in the various tissues of pea roots. This development was fastest in the phloem, first detected 1 minute post-wounding and complete by 3 hours. In some parenchyma cells full development was delayed by 20 hours. By 100 hours, wound callose was no longer detectable in parenchyma cells but remained undiminished in the phloem. The implications of these results for all studies involving callose localization are discussed.

Helminth Parasitism in Some Central Arizona Lizards

Helminth Parasitism in Some Central Arizona Lizards

A Numerical Index to Assess Early Inflorescence Development in Wheat1

Understanding of environmental effects on floral initiation and spikelet differentiation in winter wheat requires a quantitative method of assessing inflorescence development. This note presents a numerical staging scheme for measuring progression of inflorescence development for material which has been preserved in formalin‐acetic acidalcohol. The assessment is based primarily on the production of successive floral appendages; but, since these are difficult to observe in preserved material, additional criteria involving spike shape and size are included. The system works for club and for common wheats, and permits clear delineation of the timing of occurrence of floral initiation and of termination of spikelet differentiation.

Presence of Stomata-like Structures in the Ligule of Agrostis gigantea Roth

As part of an investigation into the physiological role of the grass ligule, a preliminary study of selected grasses was made to assess differences and similarities of this structure which might have a bearing on its function. A number of observations were made, amongst which was the discovery of stomata-like bodies on the ligules of certain grasses. An extensive search of the available literature has found mention of this for only one grass Ammophila arenaria (L.) Link (Gluck, 1901). This worker claimed that stornata (Spaltoffnungen) were only found on the ligule of marram of the grasses studied but gave no photomicrograph or drawing as evidence. In view of the apparent lack of information on this subject a short account of work with Agrostis gigantea Roth is presented here. Culms of the grass were collected from a ruderal habitat in Durham City during the summer of 1981 ; ligule regions were excised and stored in formalin-acetic acid-alcohol (FAA) until examined. Under the light microscope with straight FAA-fixed material little detail of the ligule surface could be determined, but after clearing in 75 per cent lactic acid (Simpson, 1929), the tissue became almost transparent and the detail readily seen. The light micrograph [Fig. 1(a)] shows the abaxial surface of the ligule (which faces the adaxial surface of the blade) and demonstrates its spinous nature as previously reported by Philipson (1937) and Davies (1959). The epidermis was composed of long cells (Philipson, 1937) with wavy, possibly pitted, walls arranged in rows parallel to the long axis, interrupted at intervals by unicellular prickle hairs and short cells (some possibly as paired silica-cork cells). Veins were found in the margins of the ligule and on a few occasions a single vein was seen running down the middle. The darker diagonal band in Fig. 1(a) is such a median vein with associated stomata-like bodies in a single row on either side of it. The marginal veins arose directly (without splitting to give a blade branch) from the marginal sheath vein while the median vein arose directly from a median sheath vein. Both types of vein appeared to stop short of the tip of the ligule and the stomata-like bodies were seen only part way along their lengths, never to the ends.

Anatomical Studies of the Neotropical Cyatheaceae. II. Metaxya and Lophosoria

The classification of the monotypic tree fern genera Metaxya and Lophosoria has undergone several revisions. Early taxonomists placed these genera, with the dicksonioid group, in the family Cyatheaceae (Diels, 1902; Christensen, 1906; Maxon, 1912). Bower (1926) stressed the importance of soral position and placed these primitive genera in the family Protocyatheaceae. Later workers (Christensen, 1938; Copeland, 1947) included the two genera in the Cyatheaceae, but excluded the dicksonioid group from that family. Based upon morphological, developmental and anatomical data, Holttum and Sen (1961) again placed Metaxya and Lophosoria and the dicksonioid ferns into the single family Cyatheaceae. More recently, Tryon (1970) revised the classification of the Cyatheaceae, excluding members of the Dicksoniaceae. He recognized Metaxya and Lophosoria as distinctive genera and placed them in an independent position at the base of his phyletic scheme for the family. In this study, the anatomy of Metaxya rostrata (H.B.K.) Presl and Lophosoria quadripinnata (Gmel.) C. Chr. is elucidated and an attempt is made to determine whether the proposed phyletic relationship between these two taxa and the squamate genera of tree ferns is supported by anatomical data. Developing shoot tips of M. rostrata and L. quadripinnata were collected in montane and lowland rainforests in Costa Rica. The plant materials were killed and fixed in formalin-acetic acid-alcohol (FAA) and sectioned on a macrotome (Lucansky, 1976a). The slices were partitioned into manageable sizes, dehydrated in a tertiary-butyl alcohol series and embedded in paraffin (Johansen, 1940). Sections (10 f(m) were made and stained in a safranin-fast green series. Stained sections were photographed with a Nikon M-35S camera, whereas entire slices were photographed with a Pentax 35 mm single lens reflex camera. Voucher specimens are deposited in the herbarium of Duke University.

Parasites of the bald eagle (Haliaeetus leucocephalus) of North America.

Information concerning parasites of the bald eagle (Haliaeetus leucocephalus) in North America is relatively sparse. Since the bald eagle in parts of the United States is an endangered species, collection of eagles for parasito!ogical study is difficult. There have been several singular reports of parasites found in oron bald eagles in the literature. However, Kocan and Locke (1974, J. Wild!. I)is. 10: 8-10) reported the first survey of helminths collected from dead and moribund bald eagles they examined from 1963-1971. Since dead and moribund eagles are collected and submitted to the National Wildlife Health Laboratory (NWHL) for necropsy to determine factors that con tribute to their mortality, the opportunity was provided to collect and identify en doparasites and ectoparasites observed during routine necropsy. This paper summarizes the parasites found in bald eagles examined from 1979-1981. Parasite specimens were obtained from a total of 84 bald eagles collected from 18 states. All helminth parasites were fixed in AFA (alcohol-formalinacetic acid). Trematodes and acanthocephala were stained with Semichon’s carmine and mounted. Nematodes were cleared in glycerine. Ectoparasites were fixed in 70% alcohol and cleared in 1014 KOH before being mounted. Wet smears made from intestinal scrapings were examined at 140 and 1000X magnification to detect the presence of protozoan parasites. Bald eagle specimens submitted to NWHL were not used exclusively for parasitological examinations, thus metazoan parasite specimens were collected by sight. Fine-mesh screens were not used. Parasites recovered from bald eagle hosts are shown in Table 1. One protozoan, two genera of Trematoda, one genus of Acanthocephala, seven genera of Nematoda, and three genera of Mallophaga were reported. New host records are indicated in the table by an asterisk. Female Contracaecum found without males or those that were found as larval stages were identified only to

Anatomical Studies of the Neotropical Cyatheaceae. I. Alsophila and Nephelea

The taxonomic treatment of the Cyatheaceae has undergone numerous revisions as summarized in previous studies (Tryon, 1970; Gastony, 1973; Lucansky, 1974). Earlier research has shown the taxonomic importance of anatomy and morphology to the study of this group of ferns (Holttum & Sen, 1961; Sen, 1964; Lucansky, 1974; Lucansky & White, 1974). Yet, despite this renewal of interest in the tree ferns, comparative anatomical data are almost totally lacking for the neotropical species. Tryon's (1970) revised classification of the Cyatheaceae recognizes three basic evolutionary lines among the squamate genera, based upon petiole scale characters. The genera Alsophila and Nephelea, with structurally marginate petiole scales having dark apical setae, constitute one evolutionary line. Similarities in the sporogenetic pattern, spore morphology, basal pinna structure, and potential for squaminate spine development also demonstrate a close phyletic relationship between these two genera (Gastony, 1973, p. 83). An attempt is made to determine whether the proposed phyletic relationship betweenA Isophila and Nephelea is supported by anatomical data. Our knowledge of anatomical data for the New World tree ferns is also increased. The following species were used in this study: Alsophila salvinii Hook., A. engelii Tryon, Nephelea erinacea (Karst.) Tryon var. erinacea, and N. polystichoides (Christ) Tryon. Voucher specimens are on file in the herbarium of Duke University. Developing shoot tips were collected in the moist, tropical, mountainous regions of Costa Rica and Venezuela. The plant materials were killed and fixed in formalin-acetic acid-alcohol (FAA) and sectioned on a macrotome (Lucansky, 1976b). The sections (slices) were partitioned into manageable sizes, dehydrated in a tertiary-butyl alcohol series, and embedded in paraffin (Johansen, 1940). Sections (8 um) were made and stained with safranin-fast green. Parts of stained sections were photographed with a 35 mm Zeiss C35 camera, whereas entire sections (slices) were photographed with a 35 mm single-lens reflex camera.

Trichuris bradleyi sp. n., a Whipworm from Octodon degus in Chile

Eight of 27 rodents, Octodon degus, collected 30 km southwest of Santiago, Chile, harbored Trichuris bradleyi sp. n. These whipworms lack a true spicular tube and the spicule is contained within the cloaca, thus differentiating the species from all others described from rodents. The species shows some similarity to T. sylvilagi Tiner 1950, especially in the length of the spicule and shape of the cloacal tube. Between July-August 1974, in the area of "Quebrada de la Plata," approximately 30 km southwest of Santiago, Chile, a biological survey was undertaken. The collection location is mountainous with steep slopes (30 to 50% grades and approximately 500-m elevation) covered by a coarse colluvial soil. The prevailing vegetation is savannah dominated by Acacia caven, although significant differences are apparent due to slope exposure. For example, north-facing slopes are covered with xerophytic vegetation (thorny shrub and Puya trichocereus association), whereas south-facing slopes are covered by a mosaic of xerophytic forest which has been degraded by human activity. The mean yearly temperature and rainfall is 14.5 C and 400 mm, respectively (Di Castri, 1968). MATERIALS AND METHODS Twenty-seven degu rodents, Octodon degus, were collected. The animals were obtained using snap-traps with oats as bait. Helminths were obtained, with some modification, employing the method described by Tagle-Villarroel (1956). They were stored in alcohol-formalin-acetic acid (AFA) solution and all nematodes were studied after clearing in lactophenol.

Male Sterility Caused by Cooling Treatment at the Young Microspore Stage in Rice Plants : IX. Revision of the classification and terminology of pollen developmental stages

Following Kihara and Hirayoshi4) we have adopted the shrinkage of FAA-fixed microspores as a criterion of classifying the microspore stage. In the present study the possibility that the shrinkage was induced by the fixatives was examined by using three fixatives: acetic acid-alcohol (AA), formalin-acetic acid-alcohol (FAA) and glutaraldehyde (GA). AA and FAA shrank microspores remarkably, while GA did not. This result shows that shrunk configurations of microspores are artifacts induced by the fixatives (AA and FAA) and led us, to revise the terminology of classifying the microspore stage. Hereafter we divide the microspore stage according to the developments of both vacuolcs and exines into three phases (early microspore phase, middle microspore phase and late microspore phase) and divide the pollen stage according to the engorgement of reserve substances into two phases (vacuole pollen phase and engorged pollen phase).

Autoradiographic Studies on the Reproductive System of Philophthalmus hegeneri Penner and Fried, 1963

Adult Philophthalmus hegeneri exposed in vitro to 3H-thymidine showed incorporation of the isotope on autoradiograms over nuclei of actively dividing cells of the testes, ovary, vitellaria, and developing miracidia within eggs. Autoradiograms of worms exposed for 1 hr to 3H-tyrosine showed heavy labeling over the ovary, vitelline cells, and the testes. Timing studies utilizing transplanted thymidine-labeled worms indicated it takes 12 days for oogonial cells to become labeled, migrate through the ovary, and be incorporated into eggs. It took 96 hr for labeled vitelline cells to reach the vitelline reservoir. Spermatogonia labeled in the initial 6-hr exposure took 48 hr to become primary spermatocytes, 72 hr to become secondary spermatocytes, 96 hr to become spermatids, 120 hr to become mature sperm in bundles, and 156 hr to reach the seminal vesicle. Studies with tyrosine-labeled worms transplanted to the host for 5 days indicated adults taken from multiple infections will not self-inseminate when isolated. Similar worms transplanted with unlabeled worms exclusively cross-inseminate. In a study on an eye fluke of birds, Philophthalmus megalurus, utilizing techniques of radioisotopic labeling, autoradiography, and transplantation, Nollen (1968a) was able to study several aspects of reproductive physiology and behavior. Timing of stages of gametogenesis and migration of vitelline cells as well as patterns of sperm transfer in single and multiple infections were determined in this study.Nollen found these flukes can selfinseminate in isolation but preferentially crossinseminate in multiple infections. Philophthalmus hegeneri, an eye fluke of shore birds in Florida, differs in several ways from P. megalurus. Among these are the apparent lack of self-insemination when adult P. hegeneri are grown in isolation (Fried, 1962). The ability of the snail intermediate host, Batillaria minima, to survive long periods out of the water makes it ideal for shipping. Thus we have been able to raise adult worms in chicks from larval stages shed by snails collected in Florida. The techniques utilized by Nollen (1968b) were applied to a study of P. hegeneri to see if species differences are apparent in reproductive physiology and behavior when compared to P. megalurus. MATERIALS AND METHODS Philophthalmus hegeneri was obtained from naturally infected Batillaria minima collected from Received for publication 2 March 1973. * Supported by the United States-Japan Cooperative Medical Sciences Program administered by NIAID: Grant Al 09330. Clearwater Harbor, Largo, Florida. Cercariae shed from snails encysted in finger bowls and were stored in aerated seawater not longer than 4 days before use. Metacercariae were induced to excyst by replacing seawater with warm (40 C) saline (0.85%), and day-old chicks (Big H Foods, Fairbury, Ill.) were infected by pipetting the metacercariae directly into the orbit. Worms were removed from the conjunctival sacs of chicks after 35 to 45 days of growth and exposed at 38 to 39 C in small stender dishes to tritiated compounds (Amersham/Searle Inc.) diluted with HedonFleig's saline. The radioisotopes used were thymidine (methyl-3H; 22.4 Ci/mmole) at 75 ,uCi/ml and L-tyrosine (-3,5-T; 1 Ci/mmole) at 100 iCi/ml. Worms were exposed to 3H-thymidine for periods of 6 hr or more to determine the time needed for optimum labeling of the cells of the reproductive system. Similar exposures were carried out with 3H-tyrosine to determine areas of incorporation in the tissues of P. hegeneri. After exposure, worms were either killed immediately in alcohol-formalin-acetic acid (AFA) or returned to the conjunctival sacs of uninfected chicks for varying periods of time. Techniques for autoradiography and transplantation of the exposed worms are described by Nollen (1968b).

The dictyate oocyte of Microtus montanus.

The resumption of mammalian oocyte nuclear maturation following oocyte growth has been typically defined as: (1) the first change in oocyte morphology after the completion of the single `resting' dictyate stage, and/or (2) changes in the oocyte initiated at the time of ovulatory stimulation. It seemed of interest to report striking changes in the dictyate oocyte which were associated with follicle development rather than ovulatory stimulation in the field vole, Microtus montanus. Ten mature (open vagina) virgin females were selected from a breeding colony of M. montanus. One ovary from each female was fixed in alcoholformalin-acetic acid, embedded in Tissue-mat and serially sectioned at 10 μm. Sections were stained with Harris' haematoxylin and eosin for general morphology and Schiff's reagent and fast green (Feulgen) for chromosomal studies. Only oocytes contained within normal follicles (absence of

Some Helminth Parasites of Panamanian Primates

Helminths were recovered from 235 (57%) of 412 Panamanian primates. Five species of trematodes, three species of cestodes, 14 species of nematodes, and three species of acanthocephalans were found. Seventeen new host records and 15 new locality records are reported. Spirura tamarini Cosgrove, Nelson & Jones, 1963, is regarded as a synonym of S. guianensis (Ortlepp, 1924). Zonorchis goliath Travassos, 1945, and Echinostoma aphylactum Dietz, 1909, are reported from primates for the first time and Parabronema bonnei (van Thiel, 1925) Diaz-Ungria, 1965, and Controrchis biliophilus Price, 1928, are reported from primates for the third time. Although helminths have been reported from Panamanian primates (McCoy, 1936; Faust, 1935; Caballero et al., 1952), apparently no general survey for these parasites has been made previously in Panama. The present study, while not exhaustive, is a record of helminths found in primates maintained in the laboratory varying lengths of time for malarial studies. From August 1965 to May 1967 helminths were recovered from 235 (57%) of 412 primates autopsied. Table I shows the species distribution of the primates examined, the numbers examined, and the numbers positive. MATERIALS AND METHODS Primates were purchased at the laboratory from native collectors. When an animal died, it was autopsied to determine the cause of death. First, the skin was removed and filarial worms were searched for in the subcutaneous tissues. Next, the internal organs were removed, placed individually in dishes of physiological saline, and either incised or teased apart. The intestinal tract, on being opened, was cut into sections and washed in saline. Any helminths seen were preserved for subsequent study. Trematodes and cestodes were killed with gentle heat, fixed in alcoholformalin-acetic acid solution, stained with Mayer's carmalum, and cleared in methyl salicylate. Acanthocephalans were killed in freshwater in order to make the proboscis protrude, preserved in 70% alcohol, and stained in the same manner as the trematodes and cestodes. Nematodes were killed and preserved in 70% alcohol. They were then studied in lacto-phenol solution and mounted in polyvinyl alcohol containing lacto-phenol. Face-on views of the nematodes were obtained by mounting the heads in glycerine jelly.

The use of papain in clearing plant tissues for whole mounts.

Materials killed and fixed in FAA (formalin-acetic acid-alcohol) and similar fixatives frequently are difficult to clear for whole mounts because the denatured proteins will not become soluble in NaOH and other clearing agents. If tissues are washed for 3 days in running water, then incubated at 40 C for 5-7 days in 2% papain buffered to pH 7.2 and activated with 15 ml of. 02 M Na2S, cell contents are partly digested. Normal clearing with 5-10% NaOH followed by chloral hydrate (sat. aq.) can then effect complete solublility of cell contents and their removal. Permanent slides can be made after staining (1% safranin O in 50% alcohol for 12 hr is successful), by dehydration through alcohols, clearing in xylene, and mounting in resin.

Pentastomida from Reptiles of Lan Yu Island (Taiwan), with a Description of Raillietiella hebitihamata n. sp.

All hosts were taken alive or were killed in the field just a few hours prior to examination. Each organ system was examined separately under a dissecting microscope. Pentastomids were killed in hot water then fixed immediately in FAA (formalin-acetic acid-alcohol). After 12 to 15 hours of fixation, specimens were washed and stored in 70 percent alcohol plus 2 percent glycerine and later studied without staining. All measurements are reported in millimeters.

THE CELLULAR PROEMBRYO OF ZAMIA AND ITS CAP CELLS

My experience confirms that of Webber (1901) and of Coulter and Chamberlain (1903), all of whom report that cones of Zamia continue their development for some time (10 days to 2 weeks) after removal from the plant. Webber (1901) was able on several occasions to check the fixation of material shipped and then killed (the interval being several days) with that fixed immediately in the field. He stated that he was unable to find any perceptible differences. Manv hundreds of proembryos in various stages of development were dissected out of the living seeds before being killed and fixed. Even stages in the e,arly development of the proembryo have been obtained without indication of damage. In these early stages the presence of a firm and tenacious archegonial membrane makes such dissections possible. However. in securing most of the very young stages for study the general procedure was to cut out and include a small cube of the surrounding gametophyte tissue. The employment of dissection permitted individual proembryos, or groups of them, to be studied and photographed as whole units; and as a further advantage m,ade it possible to orient and section them accurately. Various 'killing agents were used: Allen-Bouin Type II; Craf III and IV; formalin-acetic acidalcohol; and formalin-propionic acid-alcohol. The usual methods of embedding, sectioning and staining were employed. There are differences of opinion concerning the number of species of Zamia native to Florida. Webber (1901), who studied the plants in the field as well as in several herb-aria, held that there are two