Heidenhain's Iron-hematoxylin Research Articles

Mitochondria Load Degree in Hepatocytes of the Classical Hepatic Lobules in Chinchilla (Chinchilla lanigera)

Hepatocytes represent the majority of the liver cell population and are arranged in the form of cords placed in intimate contact with the sinusoidal capillaries. The functional complexity corroborated with the intensity of the activity of hepatocytes requires large amounts of energy. The organelles involved in the production of chemical energy used in the activity of hepatocytes are the mitochondria. The purpose of this study was to verify the mitochondrial load of hepatocytes in all areas of the classical hepatic lobules, in order to indirectly assess the intensity of hepatocyte activity in each area. Materials and Methods Five fresh corpses of chinchilla (Chinchilla lanigera) from an independent breeder from Bistrița-Năsăud county were used. Liver fragments were harvested and fixated in Kolster’s solution for 24 hours, stained with Heidenhain ferric hematoxylin, and assessed using Olympus BX41 microscope. Fixation with Kolster's solution and the staining with Heidenhain's iron hematoxylin clearly shows the hepatocytic mitochondria in shades from gray to black. The liver lobules displayed an uneven distribution of mitochondria depending on the area. In zone 1 of the classical hepatic lobule, the degree of loading of hepatocytes with mitochondria is larger than in zone 2 and much larger than in zone 3. Morphological features of the hepatocytes, including the number and distribution of mitochondria in the hepatic lobules, should improve the understanding of the physiology and pathology of the liver.

Rosuvastatin protects against coronary microembolization-induced cardiac injury via inhibiting NLRP3 inflammasome-dependent pyroptosis

Abstract Background Coronary microembolization (CME), a common reason for periprocedural myocardial infarction (PMI) after coronary interventions, bears very important prognostic implications. However, the molecular mechanisms related to CME remain largely elusive. Statins have been shown to prevent PMI, but the underlying mechanism has not been identified. Purpose To determine whether NLRP3 inflammasome contributes to CME-induced cardiac injury and to investigate the effect of statin therapy on CME. Methods In vivo study, CME models were established in C57BL/6J mice by injecting 500,000 polyethylene microspheres (diameter 9 μm) into the left ventricle chamber with the occlusion of the ascending aorta. Mice with CME were treated with 40 mg/kg/d rosuvastatin orally or a selective NLRP3 inflammasome inhibitor MCC950 intraperitoneally (20 mg/kg/d). Cardiac function was evaluated by echocardiography. Cardiac histological analysis was performed. The NLRP3 inflammasome and pyroptosis-related genes and proteins were detected. Cardiac injury was evaluated by Heidenhain's iron hematoxylin stain and serum lactate dehydrogenase (LDH) measurement. Cardiac reactive oxygen species (ROS) production and mitochondrial morphology were also determined. In vitro study, H9c2 myoblasts, stimulated with the classic NLRP3 inflammasome activators lipopolysaccharide (LPS) plus nigericin (Nig), were used to reveal underlying mechanisms of the rosuvastatin's effects. Results The expression of NLRP3, caspase-1 and interleukin-1β (IL-1β) were all increased and peaking at 3 days after CME. Mice treated with MCC950 and rosuvastatin showed improved cardiac contractile function and morphological changes, diminished fibrosis and microinfarct size, and reduced serum LDH level. Mechanistically, rosuvastatin decreased the expression of NLRP3, caspase-1, IL-1β and the pyroptosis executor Gasdermin D. Furthermore, reduced ROS level and alleviated mitochondrial damage were observed in rosuvastatin-treated mice. In vitro study, rosuvastatin effectively inhibited the activation of NLRP3 inflammasome induced by LPS and Nig in H9c2 cells, as evidenced by decreased expression of NLRP3, caspase-1, IL-1β and Gasdermin D. Flow cytometry revealed that rosuvastatin rescued LPS and Nig-induced caspase-1 activation from 22.2±1.6% to 4.09±0.85%. Meanwhile, the pyroptosis was also suppressed by rosuvastatin, indicated by the increased cell viability, decreased LDH and propidium iodide uptake. Similarly, rosuvastatin also reduced the level of ROS generation in vitro. Conclusions NLRP3 inflammasome-dependent cardiac pyroptosis plays an important role in CME-induced cardiac injury and its inhibitor exerts cardioprotective effect following CME. We also uncover the anti-pyroptosis role of rosuvastatin in CME, which is associated with regulating mitochondrial ROS. Funding Acknowledgement Type of funding source: None

Spironucleosis (Hexamitiasis, Hexamitosis) in the Ring-Necked Pheasant (Phasianus colchicus): Detection of Cysts and Description of Spironucleus meleagridis in Stained Smears

Trophozoites and cysts of Spironucleus (Hexamita) meleagridis were detected in the intestinal fluid and mucus of pheasant poults with spironudeosis (hexamitiasis, hexamitosis) following staining with Heidenhain iron hematoxylin (HIH) and the Romanowsky-type stain Hemacolor. Their morphology was consistent with that of flagellates of the genus Spironucleus, and bright-field morphologic observations were confirmed by transmission electron microscopy. Cysts occurred mostly within intestinal mucus, which was firmly compressed between microscope slides prior to staining. The internal structures of cysts were similar to those of trophozoites, allowing them to be confidently recognized. Hemacolor provided differential color staining of trophozoites and cysts, allowing accurate identification of S. meleagridis life cycle stages, even in smears in which there was heavy background staining. While HIH often produced dearer and more detailed staining of protozoan structures, in the context of a diagnostic laboratory its use was outweighed by the ease of use, rapidity of results, and differential color staining provided by Hemacolor. The possible significance of a resistant cystic stage in the life cyde of S. meleagridis is discussed.

Thyroid hormone regulation of granular intercalated duct cells in the submandibular glands of female mice.

Intercalated ducts (IDs) in the submandibular glands (SMGs) of mice exhibit a sexual dimorphism, in which a few cells in the IDs of females, but not of males, possess secretory granules. The effects of a hypophysectomy (Hypox) followed by the administration of triiodo- l-thyronine (T3) on such granular intercalated duct (GID) cells in the female gland were histologically examined. Semithin sections stained with Heidenhain's iron hematoxylin revealed that Hypox resulted in the complete disappearance of the GID cells. In Hypox females, electron-microscopy examination of the ID cells whose localization corresponded to that of the GID cells in normal females showed that these cells had a pale, centric nucleus, poorly developed rough-surfaced endoplasmic reticulum (RER) and Golgi apparatus, and no secretory granules. When T3 (1 mg/kg body weight) was given to Hypox female mice every other day for 2 weeks, the GID cells reappeared in most of the ID segments. Electron microscopy revealed that these cells had abundant secretory granules in their apical cytoplasm, a nucleus located near the base of the cell, and layered cisterna of RER and segments of Golgi apparatus in the perinuclear cytoplasm. The localization and distribution of the GID cells in the T3-treated Hypox females were almost the same as those in normal females. Taken together, these results suggest that thyroid hormones upregulate the GID phenotype, and that thyroid hormones are essential for the exocrine activities of GID cells.

Staining of intestinal protozoa with Heidenhain's iron hematoxylin.

Due to its unique properties, iron hematoxylin has been traditionally recommended for staining intestinal protozoa. This process can be simplified by reducing the number of steps and periods of permanence of the slides in some of the liquids used, without detriment to the quality of the results. Thus iron hematoxylin becomes adequate for routine use. Hematoxylin is a natural dye extracted from Haematoxylon campechianum, of the family Leguminosae. It must first be 'ripened', i.e. oxidized to hematein, which reacts with ferric ammonium sulphate to produce the ferric lake (iron hematoxylin), a basic dye. Iron hematoxylin most frequently stains regressively, i.e. the slides are first overstained and then differentiated.

Morphologic changes in the granular convoluted tubule cells of the mouse submandibular gland following hypophysectomy and hormonal replacement.

Morphological changes in the granular convoluted tubule (GCT) cells of the male mouse submandibular gland (SMG) were examined following hypophysectomy and hormonal replacement. Semithin sections stained with Heidenhain's iron hematoxylin showed that hypophysectomy severely regressed the GCT phenotype. Although only a few dispersed cells containing secretory granules were observed in the GCT segments under a light microscope, electron microscopy revealed that many regressed cells continued to constitutively elaborate apical secretory granules (although they were very small) and contained a euchromatic nucleus at the center of the cell, poor rough endoplasmic reticulum (RER) and Golgi apparatus in the perinuclear region, and well-developed basal infoldings. These findings suggest that hypophysectomy resulted in atrophy of GCT cells, but that they retained evidence of being secretory cells. 5 alpha-Dihydrotestosterone (DHT); 3,5,3'-triiodo- l-thyronine (T(3)); and dexamethasone (Dex) each enhanced the GCT phenotype of hypophysectomized males to some degree. Combined hormonal replacement with DHT + T(3) in hypophysectomized males restored a nearly normal male GCT phenotype with a full complement of secretory granules and rare basal infoldings, whereas T(3) alone induced a normal female-like GCT phenotype, with considerably abundant secretory granules and the usual short basal infoldings, in hypophysectomized male glands. Furthermore, Dex was found to synergistically enlarge secretory granules when administered with T(3) and/or DHT, although it was only weakly effective in enhancing the GCT phenotype when used alone. Taken together, the above findings confirmed that the GCT phenotype of the mouse SMG is regulated by the synergistic action of pituitary-dependent hormones.

Identification of Cryptosporidium spp. oocysts in fecal smears stained with Heidenhain's iron hematoxylin.

There is no paucity of methods for diagnosing Cryptosporidium spp. infection. The merits of immunoassays notwithstanding, microscopic identification of Cryptosporidium spp. oocysts in fecal samples remains an important diagnostic procedure. It owes the persistence of its use to such characteristics as dispensing with expensive equipment and kits, requiring only basic laboratory facilities, and having a low probability of false positive results when permanent slides are prepared, which can be re-examined in case of doubt. Cryptosporidium spp. oocysts can be readily identified in fecal smears prepared according to a regressive iron hematoxylin staining technique. The number of steps and their duration, as well as costs, were reduced to a minimum without loss of image quality and permanence of the preparations.

Ultrastructural study of hormonally responsive striated duct cells in the mouse sublingual gland.

In semithin sections stained with Heidenhain's iron hematoxylin, a few scattered granular cells were observed in the striated ducts (SDs) of sublingual glands (SLGs) of the mouse; they were seen normally only in the glands of adult males. However, it was shown by electron microscopy that many SD cells, other than these granular cells, had apical secretory granules, thus forming a granular striated tubule (named the GST in this study) in a portion of SD segments in both sexes. Sublingual GST cells had very small dense secretory granules near the apical surface, with the nucleus in the apical one-third to one-half of the cell; small Golgi apparatus; sparse rough endoplasmic reticulum (RER); and well-developed basal infoldings. However, some granular cells in male GSTs had abundant large dense secretory granules in the apical two-thirds of the cell, a basal nucleus, and modest basal infoldings. Such granular SD cells disappeared after castration in males. Granular SD cells could be induced in the GSTs of females by the injection of 5 alpha-dihydrotestosterone (DHT), triiodothyronine (T(3)), and/or dexamethasone (Dex); given simultaneously, these hormones acted synergistically in this induction. These results indicate a close similarity between the duct systems of the SLG and those of the submandibulan gland (SMG) of the mouse: granular SD cells of the GST in the SLG resemble GCT cells in the SMG in expressing some of the same biologically active polypeptides, in being sexually dimorphic, and in being under the same multihormonal regulation.

Morphologic aspects of Tetratrichomonas didelphidis isolated from opossums Didelphis marsupialis and Lutreolina crassicaudata.

Tetratrichomonas didelphidis (Hegner & Ratcliffe, 1927) Andersen & Reilly, 1965 is a flagellate protozoan found in the intestine, cecum, and colon of Didelphis marsupialis. The parasitic protozoa used in this study was found and isolated in the intestine of opossums in Pavlova starch-containing medium in Florianópolis, State of Santa Catarina, Brazil, from D. marsupialis and Lutreolina crassicaudata. The strains were cultivated in Diamond medium without maltose and with starch solution, pH 7.5 at 28 degrees C. The specimens were stained by the Giemsa method and Heidenhain's iron hematoxylin. The light microscopy study of the trophozoites revealed the same morphologic characteristics as specimens previously described.

Ameboflagellates in Bronchial Asthma

To distinguish between the existence of detached ciliary tufts (DCTs) and the possibility of protozoa in the sputum of asthma patients. One hundred six samples of sputum obtained from 97 patients hospitalized with either asthma or other respiratory diseases were examined blindly. The combination of such criteria as movement, absence of basal plate, existence of red granules, positivity for ultraviolet light and Heidenhain's iron hematoxylin stain (for protozoa) was employed to distinguish between DCTs and true protozoa. The presence of ameboflagellates in sputum has a diagnostic accuracy of 86% in predicting or ruling. out the possibility of acute asthma. The presence of ameboflagellate forms is closely related to the existence of bronchial asthma, and these result reflect some etiopathogenic significance in asthma.

A New Species of Lagenophrys (Ciliophora: Peritrichia) Symbiotic on Marine Amphipods

Lagenophrys crutchfieldi n. sp., a loricate peritrich, is an ectocommensal on two species of the marine amphipod genus Parhyale and becomes one of the few species in its genus known to occur on marine hosts. Differences in the distribution of L. crutchfieldi on the bodies of its two hosts may be linked to a difference in the force of their respiratory currents, which carry food particles to the ciliates. L. crutchfieldi is one of a small group of species in its genus hosted by talitroid amphipods, but there is no indication that members of this group have a close phylogenetic relationship. The 54 species in the peritrich genus Lagenophrys live as ectocommensals on crustaceans in many types of habitats throughout the world (Clamp, 1991); however, only five of them occur on marine or estuarine hosts. Lagenophrys eupagurus Kellicott, 1893 and the closely related L. callinectes Couch, 1967 are found on marine and estuarine decapods (Clamp, 1989; Couch, 1967), but the former species also is widespread on freshwater decapods (Clamp, 1989). Both L. cochinensis Santhakumari, 1976 and L. limnoria Clamp, 1988 are symbionts of wood-boring isopods (Clamp, 1988a; Santhakumari, 1976; Santhakumari & Gopalan, 1980; Santhakumari & Nair, 1985). Lagenophrys tattersalli Willis, 1942 occurs on the gills of marine amphipods in Great Britain (Willis, 1942) and France (Clamp, unpublished observations). Also, Fenchel (1965) reported an undescribed species of Lagenophrys from marine amphipods collected in the coastal waters of Denmark. I have discovered and describe herein an additional marine species of Lagenophrys on two members of the amphipod genus Parhyale Stebbing, 1897, whose species are widely distributed in tropical and subtropical environments (Barnard, 1979; Shoemaker, 1956). MATERIALS AND METHODS Specimens of Parhyale hawaiensis (Dana, 1853) were collected with a dip net. They were fixed in Bouin's fluid for 24 h and transferred to 70% ethyl alcohol for preservation. Samples of L. crutchfieldi also were obtained from specimens of P. hawaiensis and P. penicillata Shoemaker, 1956 in the crustacean collection of the National Museum of Natural History, Smithsonian Institution (herein referred to as NMNH-CC). Permanent preparations were made by staining with Heidenhain's iron hematoxylin and protargol. Copper was omitted from the protargol solution. Some I I thank Dr. Austin B. Williams for arranging my visit to the crustacean collection of the National Museum of Natural History and Dave Penrose, who helped me collect amphipods on a memorable trip to Florida. Publication costs, in part, are being met by a grant from the Spencer-Tolles Fund of the American Microscopical Society. TRANS. AM. MICROSC. Soc., 112(1): 62-68. 1993. ? Copyright, 1993, by the American Microscopical Society, Inc. This content downloaded from 207.46.13.124 on Wed, 22 Jun 2016 05:21:34 UTC All use subject to http://about.jstor.org/terms VOL. 112, NO. 1, JANUARY 1993 ciliates were examined alive, but only fixed and stained material was used for drawings and measurements (see Clamp, 1990). Two samples of individuals stained with hematoxylin were measured with a filar micrometer. Only individuals oriented with their dorsal surface toward the observer and with their ventral surface parallel to the plane of the slide, or nearly so, were selected for measurement. Height of the lorica was measured by using the calibrated scale on the fine focusing knob of the microscope to determine the focal distance between the lorica's dorsal and ventral surfaces. Phase-contrast microscopy was used for this measurement because of the transparency of the lorica.

Immunoreactive copper-zinc superoxide dismutase in damaged human myocardium.

The myocardium in 50 autopsy cases was studied using immunostaining for copper-zinc superoxide dismutase (CuZn-SOD) and standard histochemical procedures. Mucinous degeneration observed in 42 cases showed moderately enhanced expression of immunoreactive CuZn-SOD in lesions which were stained strongly by periodic acid-Schiff but negative with Heidenhain iron-hematoxylin (HIH), von Kossa and luxol fast blue (LFB) stains, whereas coagulation necrosis in 4 cases revealed almost identical immunostaining for CuZn-SOD and HIH to that of contraction band necrosis, i.e. strongly positive HIH staining but negative immunostaining. Basophilic alteration of the myocardial cells in sections fixed with 4% formalin in 2% calcium acetate was seen in 29 cases, being identified frequently in isolated cells as well as in several foci varying considerably in size. This type of alteration demonstrated significantly enhanced expression of immunoreactive CuZn-SOD and was strongly positive with von Kossa and LFB stains. The present study indicates that the myocardium can be affected by free radicals produced in any organ of the body, and that subsequently, insoluble phospholipids react with calcium ions in the fixative and accumulate in the basophilic sarcoplasm.

A New Species of Lagenophrys (Ciliophora: Peritricha: Lagenophryidae) Ectocommensal on the Wood-Boring Isopod Limnoria (Flabellifera: Limnoridae)

Lagenophrys limnoriae n. sp., a loricate peritrich, occurs on the pleopods of species of the marine isopod genus Limnoria. The new species is distinguished from other members of Lagenophrys by a combination of characteristics, including proportions of the lorica, an anterior bulge in the lorica, the shape and height of the lips of the lorica aperture, the frequent presence of rows of bead-like thickenings on the lorica surface, and the shape of the macronucleus. L. limnoriae probably benefits from food particles carried in the current that the hosts generate to ventilate their burrows. The genus Lagenophrys Stein, 1852 is a cosmopolitan group of loricate peritrichs that contains more than 50 known species currently. Members of Lagenophrys3 are obligate ectocommensals of crustaceans and apparently depend on currents created by respiratory or locomotory activities of the host to bring them particulate food (Clamp, 1973), which then is captured by suspension feeding as in other peritrichs. Amphipods, decapods, and isopods are the most frequent hosts, although some species of Lagenophrys utilize copepods, ostracods, and cladocerans. Few members of the genus occur on marine hosts, only one of them on marine isopods (Santhakumari, 1976; Santhakumari & Gopalan, 1980). In addition, an undescribed species of Lagenophrys was observed on the marine wood-boring isopod Limnoria Leach, 1813 by Mohr (1959). I discovered and describe herein a new species of Lagenophrys occurring on two species of Limnoria. This new species appears to be the one observed by Mohr (1959). MATERIALS AND METHODS Specimens of Limnoria were collected by hand from pilings and wooden debris in the intertidal zone at localities in North Carolina and British Columbia. I am grateful to Drs. Phyllis Bradbury and Barbara Grimes for reading of the manuscript. This paper was part of a thesis submitted as partial fulfillment of the requirements for the Ph.D. in Zoology at North Carolina State University. It is Paper No. 2 for 1987 of the North Carolina Biological Survey. 2 Reprint requests should be directed to the museum address. 3 Jankowski (1980) moved all species of Lagenophrys whose loricae are circular in outline to the new genus Circolagenophrys, leaving two species whose loricae are oval in outline in Lagenophrys. Lorica shape is not a valid generic character in the Lagenophryidae (Clamp, 1984); therefore, Lagenophrys and not Circolagenophrys is the correct generic designation of the species described in this paper. TRANS. AM. MICROSC. SOC., 107(1): 12-16. 1988. ? Copyright, 1988, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.146 on Fri, 10 Jun 2016 06:31:18 UTC All use subject to http://about.jstor.org/terms VOL. 107, NO. 1, JANUARY 1988 They were fixed in Bouin's fluid for at least 24 h and then transferred to 70% ethyl alcohol. Permanent preparations were made by staining with Heidenhain's iron hematoxylin (Kirby, 1950). Samples of peritrichs were stained with protargol using the basic method given in Kirby (1950), except that copper was omitted from the protargol solution. Unfortunately, the buccal infraciliature was not stained clearly enough in any individual ciliate to permit its description. Material also was fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer and examined by scanning electron microscopy (SEM) following critical-point drying and coating with carbon and gold. Fixed and stained material was used for all drawings and measurements (living Lagenophrys are of little value in taxonomic work because some critical features cannot be seen satisfactorily in the living state). Physical attributes of one sample (n = 25) were measured with a filar micrometer. Individuals drawn or measured were viewed from the dorsal aspect and were oriented parallel to the plane of the slide or nearly so. Special terminology applicable to Lagenophrys (see Kane, 1965) is used in this paper.

Light-microscopical appearance and ultrastructure of Blastocystis hominis, an intestinal parasite of man

A study on Blastocystis hominis was undertaken for the purpose of clarifying the morphology of the organism using the following techniques; Giemsa, Heidenhain Iron Hematoxylin, and DAPI stains, phase-contrast microscopy, and transmission- and scanning-electron microscopy. Microscopic examinations of the lumen fluids aspirated at the endoscopical examination revealed the habitation of B. hominis in the lower ileum and cecum of a patient. When examined light-microscopically, organisms from stool materials, cultures, and aspirated intestinal lumen contents of a patient showed morphological resemblances to each other except for variations in size. Vacuolated cells, which were spherical in shape and characteristically had a large central vacuole and a narrow rim of cytoplasm containing nuclei and some inclusions, were the only form of the organism observed in this study, although the contents of the vacuole notably varied. DAPI stains clearly revealed the nucleus and the possible mitochondrion in the narrow rim of cytoplasm. Phase-contrast microscopy of fresh materials prepared with physiological saline was recommended for diagnosis. When examined electron-microscopically, the organisms were coated with a capsule that was composed of fine filamentous materials. All the organisms contained a central vacuole although the contents of it varied considerably. The cytoplasm gave the organism a signet ring appearance and contained cristate mitochondria, a great number of ribosomes,Golgi's apparatuses, cytoplasmic microtubules, and nuclei with a nucleolus. Very few of the ultrastructures are those that would be expected of a yeast. Recent occurrences of B. hominis infection in Kyoto City, Japan, during a two-year period (June 1983 to August 1985) were also reported.

Staining of interphase nuclei and mitotic figures in cultured cells with Alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.

魚類の腎系球体傍細胞顆粒(JG顆粒)の組織学的検討

A histological investigation was made on the juxtaglomerular cell granules (JG granules), using the kidneys of rockfish, Sebastiscus marmoratus, rabbitfish, Chimaera monstrosa, and pig, Sus scrofa. JG granules in these three species were clearly stained with Bowie's method . In other staining methods, however, remarkable aifferences were observed in JG granules of these species. Only JG granules of rockfish were PAS-positive in PAS and Mayer's acid hemalum stain. In Heidenhain's iron-hematoxylin stain, JG granules of pig and rabbitfish were siderophilic. Furthermore, JG granules of porcine kidney were stained with phloxine in Gomori's chrome-hematoxylin and phloxine stain. On the other hand, in Mallory's triple stain, JG granules in porcine kidney were stained with acid fuchsin, whereas those in rockfish and rabbitfish with orange G.

Aspects of the Larval Development of Ophthalmophagus sp. (Trematoda: Cyclocoelidae)

Specimens of Ophthalmophagus (near) singularis were collected from the nasal cavities and orbits of clapper rails (Rallus longirostris) from Mississippi tidal marshes. The miracidia hatched in utero, emerged, and attached to marine snails (Melampus bidentatus and Detracia floridanus). The rediae of the miracidia bored into the snails, leaving the empty miracidia behind. By 3 weeks postexposure the rediae contained metacercariae. This is the first report on the life history of cyclocoelids in the genus Ophthalmophagus and the first demonstration of cyclocoelids parasitizing marine snails. Cyclocoelid life histories have been discussed by Taft (1973, 1975). Cyclocoelid larval development is condensed. Each miracidium contains a fully formed redia. After attachment to a snail by the miracidium, the redia penetrates. Within the pulmonate host, rediae will produce cercariae that either encyst within rediae producing them or in snail tissue. Birds ingesting gastropods with mature metacercariae become infected. The present study constitutes the first report of a life history in the genus Ophthalmophagus (Stossich 1902) and is the first record of cyclocoelids parasitizing marine snails. MATERIALS AND METHODS Gravid Ophthalmophagus (near) singularis (Stossich 1902) were collected from the nasal cavities and orbits of clapper rails (Rallus longirostris). Worms placed in dilute (10-15 %o) sea water released miracidia to which wild Littorina irrorata, Melampus bidentatus, and Detracia floridanus were exposed. Sora rails (Porzana carolina) and Virginia rails (Rallus limnicola) were additional sources of miracidia for infecting M. bidentatus. After exposure, snails were placed in plastic gallon jars containing dried marine emergent vegetation and approximately 3 cm of sea water, the level of which was maintained by adding distilled water. Snails were examined weekly and larval development noted. Infected snails or isolated rediae were fixed for sectioning in Bouin's or Zenker's solution and stained with Heidenhain's iron hematoxylin or Mallory's triple stain. Adult trematodes were fixed in AFA, stained with paracarmine and counterstained with fast green. Fifteen whole mounts and one secReceived for publication 4 October 1977. * Department of Biology and Museum of Natural History, University of Wisconsin-Stevens Point, Stevens Point, Wisconsin 54481. t Gulf Coast Research Laboratory, Ocean Springs, Mississippi 39564. + Present address: Dauphin Island Sea Lab., P.O. Box 386, Dauphin Island, Alabama 36528. tioned specimen (stained with Harris' hematoxylin) from an earlier study (Heard, 1970) also were examined and measured. All molluscs and rails were collected from tidal marshes in Jackson County, Mississippi. Measurements are in micro-

A rapid method to demonstrate muscle cross-striations with thionin.

Frequently used methods for the demonstration of cross-striations of muscle fibers are Mallory's phosphotungstic acid hematoxylin (PTAH) (Mallory 1939 and Heidenhain's iron hematoxylin (McManus and Mowry 1960). Both require long staining periods; in addition, Mallory's method specifies fixation or mordanting in Zenker's fluid. A modified Mallory's PTAH procedure has been developed to accelerate the staining of formalin fixed tissue (Bohacek 1966). Preparation of solutions and actual staining time are still lengthy. The method of Menzies (1961) using bromphenol blue, though no mordanting is required, specifies 4-24 hours staining. None of these techniques are practical when time is a major factor. This report describes a staining procedure which makes the differentiation of muscle cross-striations quick and reliable.

Application of Heidenhain's iron hematoxylin staining to epon-embedded tissues.

A method for the application of Heidenhain's iron hematoxylin staining to epon-embedded tissue sections is described.Various tissues of the mouse were fixed with glutaraldehyde followed by osmium tetroxide, embedded in epon-resin, and cut into semi-thin sections about 1μ thick. The sections were affixed to the slide surface by heating on a hot-plate.Heidenhain's iron hematoxylin solution was prepared according to ROMEIS' recipe. The staining procedure which has been utilized for paraffin and celloidin sections was applied to the epon-sections with slight modification, e. g., heating the utilized solutions. The embedding medium was not removed for the staining.The results of the present staining show that the connective tissue fibers, nuclear chromatins and nucleoli were commonly stained in dark shades of black, while the cytoplasm and nuclear sap showed light shades of yellowish brown. The keratinized layer of stratified squamous epithelium, glycogen areas of hepatic cell, goblet cell mucus, Paneth cell granules and the striated border of intestinal epithelium were selectively emphasized in dark shades. On the other hand, the sarcoplasm of muscle fibers, myelin sheaths and erythrocytes appeared in light shades. Under mounting with Canada balsam the stain remains stable for at least a year.The present method is useful especially in tissues containing a cosiderable amount of connective tissue fibers but is valid also as a general stain for all epon-embedded tissues.

Two New Myxosporida, Henneguya gambusi sp. n. and Myxosoma pharyngeus sp. n., in the Mosquitofish, Gambusia affinis (Baird and Girard)

Two new species of Myxosporida, Henneguya gambusi and Myxosoma pharyngeus, are reported from connective tissues of the integument and pharyngeal cavity, respectively, of the mosquitofish, Gambusia affinis. Immature and mature stages of the parasites are described and the nature of their infections are discussed. H. gambusi is monosporous or disporous while MA. pharyngeus is principally disporous but monosporous and polysporous development is also thought to occur. Both species are reported as pathogenic. During a survey of parasites of the mosquitofish, Gambusia affinis (Baird and Girard), from northcentral Oklahoma, two types of myxosporidan infections were found. These parasites are believed to be new species and both are herein described. The specific names H. gambusi and M. pharyngeus, referring to the name of the host and the location of cysts, respectively, are suggested. MATERIALS AND METHODS Three collections of fishes, including 143 mosquitofish, were obtained from Little Stillwater Creek, Payne County, Oklahoma, during September, October, and December 1969. Selected tissues of G. affinis were fixed in Schaudinn's, Kaformacet, or Zenker's fluids, embedded in paraffin, sectioned at 7 ,u, and stained by routine histological methods. The stains used were Delafield's hematoxylin, Heidenhain's iron-hematoxylin, and Mallory's aniline-blue connective tissue stain. Smears were prepared by transferring intact cysts through Schaudinn's fixative, Heidenhain's iron-hematoxylin, or Feulgen's nuclear reagents, alcohol, and clearing agent, followed by rupturing the cysts in a drop of mounting medium. Measurements of spores were made from fresh material in 0.65% NaCl and from fixed and stained spores that had been sectioned in situ or prepared as smears. Thirty-five fresh spores and 35 fixed and stained spores were measured for both H. gambusi and M. pharyngeus. One hundred ninety-four other fish, including 28 Lepomis megalotis, 45 L. cyanellus, 26 Carpiodes carpio, 23 Ictalurus natalis, 14 Cyprinus carpio, 9 Notemigonus crysoleucas, and 49 unidentified cyprinids and catostomids were colReceived for publication 30 April 1971. * Present address: Department of Biology, Lincoln Land Community College, Springfield, Illinois. t To whom reprint requests should be addressed. + Present address: TVA, Muscle Shoals, Alabama. lected and examined but none were found to contain parasites of the type herein described.