Alcoholic Bouin Research Articles

Sex hormones in Strombus pugilis (Mollusca: Gastropoda) in different gonadal stages

Sex hormones play an essential role in sexual differentiation, maintenance of sexual characteristics, gamete maturation, and mating behavior. However, very little is known about their dynamics in molluscs. We conducted a study on sex hormone (17β-estradiol, testosterone, and progesterone) concentrations in male and female Strombus pugilis to identify their variations at different gonadal stages. A total of 90 organisms (30 per month) were collected in February, September, and November 2016. The gonadal digestive gland complex of each specimen was dissected and divided into two sections. One section was set in alcoholic Bouin's fluid and processed with classic histological techniques; the second was macerated with 80% ethanol to extract steroids and analyzed by enzyme immunoassays. Histological section analysis was used to classify gonadal development into three stages: gametogenesis, mature, and undifferentiated. Mature females were observed in September. Testosterone and 17β-estradiol concentrations in both sexes were highest in the mature stage. In S. pugilis, 17β-estradiol, progesterone, and testosterone were all present, with higher concentrations associated with reproductive activity.

Process of Oogenesis in Proechinocephalus Egreti (A Digenetic Trematode) Shrivastava, 1960

<div><p> <em>Present paper deals with the study of process of oogenesis of a digenetic trematode Proechinocephalus egreti (a digenetic trematode) of the family Echinostomatidae was collected from the intestine of an Indian cattle egret, Bubulcus ibis coromandus. A few of parasites were flattened on a clean slide under the slight pressure of a cover glass & fixed in Alcoholic Bouin,s fluid for 12 hours. Stains like Gover’s Carmine, Mayer’s Para carmine & Haemalum were used for the preparation of whole mounts for identification & the study of female reproductive organs & the process of oogenesis in Proechinocephalus egreti. </em></p></div>

Ultramorphological characteristics of Chrysomya megacephala (Diptera, Calliphoridae) eggs and its eclosion

Chrysomya blowflies are originally from Africa and Australasia and were introduced in the American Continent, probably as a sheep parasite, in the 1970s. These flies are extremely important for medical-sanitary purposes and are useful to forensic entomology being used as an indicative of decomposition time in human corpses. The morphology of the larval and pupal stages of some species has been already studied by different authors demonstrating that it is possible to identify them in premature stages. In this study, Chrysomya megacephala egg ultramorphology was analyzed to describe its structure, generating data for further comparison between different species and genera. The blowflies were collected in Rio Claro city, SP, Brazil. Flies were attracted by fish carcasses and maintained in net cages; small portions of minced meat were placed in plastic pots for egg laying. Eggs were collected, fixed in alcoholic Bouin, prepared according to SEM routine and observed under Philips scanning electron microscope. C. megacephala eggs are oval with one flat face and another convex, measuring approximately 0.52 (±0.03) mm of length and 0.12 (±0.02) mm of width. The micropyle is circular in shape, with two perforations enclosed by countless small projections and is located in one of the extremities in the interface between the flat and convex faces. On the convex surface hexagonal imprints were observed, this surface is impermeable to water and gases. The flat surface has numerous round projections with different sizes, creating a permeable surface with a thinner chorion from which the larvae hatches. The larvae presents itself all wrinkled and with numerous cuticular projections with a thorn shape facing the posterior region. The cuticle projections with a thorn shape from C. megacephala larvae are probably a vestige of the thorns found in larvae of parasite Diptera.

Regionalization of epididymal duct and epithelium in rats and mice by automatic computer-aided morphometric analysis

To establish a rat and mouse epididymal map based on the use of the Epiquatre automatic software for histologic image analysis. Epididymides from five adult rats and five adult mice were fixed in alcoholic Bouin's fixative and embedded in paraffin. Serial longitudinal sections through the medial aspect of the organ were cut at 10 microm and stained with hematoxylin and eosin. As determined from major connective tissue septa, nine subdivisions of the rat epididymis and seven for the mouse were determined, consisting of five sub-regions in the caput (rat and mouse), one (mouse) or three (rat) in the corpus and one in the cauda (rat and mouse). Using the Epiquatre software, several tubular, luminal and epithelial morphometric parameters were evaluated. Statistical comparison of the quantitative parameters revealed regional differences (2-5 in the rat, 3-6 in the mouse, dependent on parameters) with caput regions 1 and 2 being largely distinguishable from the similar remaining caput and corpus, which were in turn recognizable from the cauda regions in both species. The use of the Epiquatre software allowed us to establish regression curves for different morphometric parameters that can permit the detection of changes in their values under different pathological or experimental conditions.

Spider Ovarioles and Their Attachment

Warren (1926) stated he observed spider oocytes free in the coelom outside the ovary between the silk glands and the gut ceca. He believed these oocytes broke their funicular connections to the ovary and that in oviposition they had to migrate through the coelom and somehow break an opening into the distal oviduct and from there pass into the uterus. Apparently he interpreted the peripheral position of oocytes iir ovarioles to mean they were unconnected to ovaries. He did not observe their thin funicular connections. Savory (1928) reported the tenuous funiculi connecting the ovarioles to the ovaries in some spiders, but said they disappeared before oviposition. He further stated that the oocytes were positioned between the intestines and the silk glands in the body, and that oocytes passed single file through a perforation near the junction of the oviduct and the uterus and then passed out of the spider's body. Sadana (1970, 1972) reported the penetration of the ovarian wall by immature oocytes, which came to lie in their maturational peripheral positions attched to the ovary by funiculi. But he disagreed with Savory as to how the oocytes exited the body. Sadana claimed that the ovarian wall became thinner and thinner until large holes appeared and the bundles of oocytes entered the ovarian lumen from the body cavity through these holes. Once in the ovary they passed out of the body through the oviducts. Seitz (1971) tested Warren's claims in his study of vitellogenesis in Cupiennis salei Keys. Seitz claimed that only after the ovariole had broken its connection with the ovary did the oocyte within undergo secondary vitellogenesis. Gowan (1985) found contrary evidence in Lycosa lenta Hentz. In his serial sections, mature oocytes were fully connected to the ovary by very thin ovariole funiculi. He was able to remove a complete ovary with ovarioles still attached from a female in midoviposition, and found a continuous stream of oocytes passing through the ovarian lumen, through the oviducts, and out of the spider's body. The authors propose that spider ovarioles do not break their funicular connections to the ovary before of after secondary vitellogenesis or oviposition. Evidence is presented in support of this view, and an explanation of earlier findings by other authors is suggested. Gravid females of Pardosa, Lycosa, and Misumena spp. were killed with chloroform and their petioli cut. Ovaries and their attached oocytes were dissected from abdomens and placed in safranin stain on slides for squashing. The abdomen from one gravid Pardosa spp. was fixed in alcoholic Bouin's, dehydrated with tetrahydrofuran, and infiltrated and embedded in Paraplast. Sections were cut at 8,um, mounted, and stained with Rapid Mallory Heidenhain polychrome stain (Humason 1972). In addition, Gowan's (1985) original serial sections of Lycosa lenta were searched for connections between mature ovarioles and ovaries. Fig. 1 was taken from these slides. In our microdissections of gravid females, oocytes were seen in ovarioles still attached to the ovaries by funiculi. These ovaries could be removed with ovarioles hanging from them. Large, mature oocytes in ovarioles were frequently so tightly packed in the

Alcoholic Bouin fixation of insect nervous systems for Bodian silver staining. I. composition of 'aged' fixative.

Alcoholic Bouin (Duboscq-Brasil) fixative being 'aged' at 60 C to improve tissue preservation and subsequent staining was sampled at various stages to determine its histological effectiveness and chemical composition. Histological performance was tested using ventral nerve cord ganglia of the cockroach Periplaneta americana and the locust Schistocerca gregaria. Chemical analysis was by ultraviolet spectroscopy, thin layer and gas-liquid chromatography, and mass spectrometry. Histological performance improved rapidly during the first 7-10 days and composition changed correspondingly. The rate of change then slowed as a more stable condition was approached. Fully aged solutions, after about 40 days, giving optimum fixation and staining, contained little more than half the amounts of the volatile components (formaldehyde, ethanol, and acetic acid) in the original mixture, together with ethyl acetate and a formal, diethoxymethane, as the principal reaction products, but picric acid content showed little change. Older ('overaged') solutions, fully aged and then kept at room temperature for 1-2 yr, gave poorer fixation and staining and contained still less of the original volatile constituents and correspondingly more of the reaction products. A 'simplified synthetic aged alcoholic Bouin' (15 ml 40% formaldehyde, 35 ml ethanol, 3.5 ml acetic acid, 5 ml ethyl acetate, 15 ml diethoxymethane, 0.46 g picric acid, and water to 100 ml) closely stimulated the performance of the fully aged orthodox fixative without the need for aging.

Alcoholic Bouin fixation of insect nervous systems for bodian silver staining. III. A shortened, single impregnation method.

Satisfactory Bodian silver staining of paraffin wax sections of both locust (Schistocerca gregaria) and cockroach (Periplaneta americana) central nerve tissue can be obtained with only one impregnation, instead of the usual two, by the following modified procedure. Freshly dissected ganglia are fixed in an improved synthetic alcoholic Bouin (40% formaldehyde 0-15: ethanol 25: acetic acid 5: picric acid 0.5: either ethyl acetate 5 and diethoxymethane 15, or ethyl acetate 25: distilled water to 100). Formaldehyde content governs intensity of glial staining (little or none without formaldehyde) and the mixture with more ethyl acetate substituted for diethoxymethane gives more intense staining overall. Sections are impregnated once only, overnight, in 2% Protargol solution brought to about pH 8.4 with ammonium hydroxide and containing 1.3 g of copper per 65 ml. Depending on fixative composition, species, section thickness and contrast desired between nerve fibers and background, the subsequent distilled water rinse is shortened or omitted and sections are developed in 1% hydroquinone with sodium sulfite content reduced (to 2.5-4% Na2SO3.7H2O) for thinner (10 micrometer) sections but normal (10%) for thicker (20 micrometer) ones. Sections are finally washed, gold intensified, treated with sodium thiosulfate and dehydrated, cleared and mounted as usual. Results are slightly lighter than with normal double impregnation but entirely suitable for studies of neuroanatomy.

Alcoholic Bouin fixation of insect nervous systems for Bodian silver staining. II. Modified solutions.

A previously devised synthetic equivalent of 'aged alcoholic Bouin (Duboscq-Brasil) fixative was modified in various ways to discover which of the chemical changes brought about by aging were important in improving fixation and staining. Effects were tested with ventral nerve cord ganglia of the cockroach Periplaneta americana, locust Schistocerca gregaria, and honey bee Apis mellifera. Formation of reaction products, chiefly ethyl acetate and diethoxymethane, seemed to play only a subsidiary role: neither individually appeared essential as long as a sufficient quantity of one or the other was present. In place of diethoxymethane, ethyl acetate concentration could be increased to 25% with little effect on results. Reduction in concentration of two of the original constituents, formaldehyde and ethanol, appeared to be the principal factor in improving fixation. Varying the concentration of each original constituent individually revealed that formaldehyde mainly increased glial staining, ethanol increased tissue shrinkage and reduced overall staining intensity, acetic acid improved preservation, and picric acid decreased glial staining but produced few other effects within a wide range of concentrations, though its omission seriously impaired overall preservation and staining. Varying the ethanol and acetic acid concentrations simultaneously confirmed that they acted in opposite ways. A decrease in ethanol and an increase in acetic acid both improved results. The optimum mixture, 'improved synthetic alcoholic Bouin' (40% formaldehyde 0-15: ethanol 25: acetic acid 5: ethyl acetate 5: diethoxymethane 15: picric acid 0.5: water to 100), gives better preservation and more intense staining, and formaldehyde content can be varied to give the degree of glial staining and more intense staining, and formaldehyde content can be varied to give the degree of glial staining required. Without formaldehyde glial staining is virtually eliminated, while preservation and staining of the neurons appears unaffected. This modification seems to offer a valuable advance in technique.

A Double Infiltration Technic for Serial Sectioning of Heavily Sclerotized Insects

A double infiltration method using n-butyl alcohol in conjunction with celloidin and paraplast plus (BCPP) is described. Serial sections cut at 4 microns thick of heavily sclerotized insect cuticle fixed with alcoholic Bouin's fluid are easily obtained. The architecture of the cuticle and the morphology of the underlying tissue show little distortion and excellent staining.

A Double Infiltration Technic for Serial Sectioning of Heavily Sclerotized Insects

A double infiltration method using n-butyl alcohol in conjunction with celloidin and paraplast plus (BCPP) is described. Serial sections cut at 4 microns thick of heavily sclerotized insect cuticle fixed with alcoholic Bouin's fluid are easily obtained. The architecture of the cuticle and the morphology of the underlying tissue show little distortion and excellent staining.

Simple fluorescence staining of insect central nerve fibres with procion yellow.

Nerve fibres in the central nervous system of the cockroach Periplaneta ameri-cana can be displayed by staining whole ganglia for 1–2 hr in a saturated solution of Procion yellow M-4R in cockroach saline diluted to maintain isotonicity. Selected fibres are stained preferentially by cutting a peripheral nerve or interganglionic connective close to the ganglion, or damaging neuron cell-bodies. The ganglion is washed in saline, fixed in alcoholic Bouin, dehydrated and embedded. Under fluorescence microscopy, sections show stained fibres brilliant yellow against a green background. The method is simpler than intracellular injection and demonstrates even the finest fibres.

Descriptive Enibryogeny of the Mygalomorph Spider Antrodiaetus unicolor1,2

Material for the study of the embryogeny of the mygalomorph spider, Antrodiaetus unicolor (Hentz) (Araneae: Antrodiaetidae), was obtained from gravid females collected in North Carolina and transferred to test tubes, in which they oviposited. The eggs were incubated at 24—26°C in paraffin oil. Stages were fixed in alcoholic Bouin's and dehydrated for whole mount and sectioned preparations. Shortly after fertilization, the yolk became arranged into columns. Between these columns, cytoplasm extended from the centroplasm to periplasm. The fertilization (vitelline) membrane, surrounded by the chorion, overlaid the periplasm. As cleavage progressed, the yolk shifted into cones while the cleavage cavity became established. Once the cleavage nuclei reached the periplasm, the uppermost portion of the yolk spheres commenced to fall onto the lower yolk portion closing the cleavage cavity. This contraction phase was completed 6 days after oviposition. During gastrulation, the primitive cumulus arose from the primitive plug and migrated to the periphery of the egg. With subsequent dissolution of the cumulus, the dorsal field widened, and the beginning protozonites began to form around the abdominal pole. At least 5 segments were visible between the cephalic and caudal lobes. With the development of the cheliceral segment, the first 2 opisthosomal segments appeared. Subsequent opisthosomal development entailed the formation of succesive segments until the maximum number of 12, including the caudal lobe, was reached. The opisthosoma normally consisted of a preabdomen and postabdomen, the latter projecting anteriorly along the ventral portion of the embryo. Both the pre- and postabdomen contributed to the abdomen of the foetus. Reversion commenced following the formation of the last opisthosomal segment and was completed within 2½ days. Hatching occurred 20½ clays following oviposition.

Protective paraffin infiltration of soft tissues in insects to facilitate softening of hard exoskeletons.

This technique can produce serial sections as thin as 5 μ from hard chitin-covered materials of insects or other arthropods. Procedures: Fix with alcoholic Bouin's fluid for 3 hr. Henceforth subject material to partial vacuum in each step to ensure a final proper embedding. Wash with 80% ethanol 2 or 3 times for 2 hr or until the picric acid is largely removed. Dehydrate to 90% ethanol and give 2 changes of n-butanol 2 hr each, and one of a 1:1 n-butanol-paraffin mixture in 56-57° oven for 12 hr. Finally, use 2 baths of pure paraffin, 3 hr each, to complete the infiltration. After the last bath, withdraw the specimen from the paraffin, and remove the superficial paraffin, first mechanically and then with a xylene bath for 4 min. Rinse first with n-butanol, and afterwards with absolute ethanol, 2 min each. The compound eyes are protected with a paraffin covering, the specimen is hydrated with a 1% aqueous solution of detergent for 1 hr and then washed with running tap water. The material is treated with a ...

Silver Staining of Insect Central Nervous Systems by the Bodian Protargol Method

Improved fixation of ganglia of the central nervous system of Periplaneta americana and Schistocerca gregaria for silver staining by Power's (1943) modification of the Bodian protargol method is given by alcoholic Bouin aged for at least 40 days at 60° C. During impregnation of sections, increased copper and decreased pH give paler staining, more selective for nerve fibres. Prolonging impregnation from 24 to 48 hours weakens the stain and decreases selectivity. The intensity of the stain depends chiefly upon the amount of unreduced (developable) silver combined with the tissues; selectivity is determined mainly by the number and distribution of the reduced silver particles (‘nuclei’). In development, increased sodium sulphite gives more differentiation, increased hydroquinone gives less. Optimum developer composition depends upon impregnation, and thick sections need more differentiation than thinner ones. Within limits, change in one of the factors that control staining can be balanced by changes in others, but by suitable adjustment of the conditions the result can be varied from almost total staining of nerve fibres, for general neuroanatomy, to highly selective staining for tracing individual fibres.

Orcein-Hematoxylin in Iodized Ferric Chloride as a Stain for Elastic Fibers, With Metanil Yellow Counterstaining

Autopsy and biopsy specimens of human skin were fixed overnight in alcoholic Bouin's solution, embedded in paraffin, cut at 7 μ, deparaffinized, hydrated to 70% alcohol, and treated as follows—stained 2 hours in a mixture consisting of: 0.2% orcein in 70% alcohol and 1% HC1 (conc.), 125 ml; 5% hematoxylin in absolute alcohol, 40 ml; 6% FeCl3 in water, 25 ml; and aqueous I2-KI (1:2:100), 25 ml—rinsed in distilled water until the excess stain was removed—differentiated in 1.2% FeCl3, 5-15 sec—washed in running water, 5 min—differentiation completed in 0.01% HC1 acid-alcohol, 1 min—a dip in 95% alcohol—distilled water, 2 min—0.25% aqueous metanil yellow, 5-10 sec—a 95% alcohol dip—dehydrated in absolute alcohol, xylene, and mounted in a resinous medium. The technic combines the orcein of Pinkus' stain and the hematoxylin mixture of Verhoeff into a single staining solution and gives sharp and reliable results for both coarse and extremely delicate elastic fibers. These stain purple; nuclei, violet; and backgr...

A Dissection Technique for Studying Internal Anatomy of Different Stadia of Noctuidae1

During studies on the internal anatomy of moths, morphologists often encounter difficulties in removing the muscles and the fat body. Routine sectioning techniques are time consuming. The alcoholic Bouin's fixative (after Drake and McEwen 1959) commonly employed in microhistological techniques renders soft tissues too brittle for dissection. There are several other difficulties involved in dissecting fresh specimens—e.g., nerves, in particular, are very difficult to see. An ideal technique would harden the muscles and soften the tracheae so that they can be easily removed. Also, the ideal fixative and staining techniques should soften the exoskeleton, cause a minimum of shrinking or swelling of the tissues, and be comparatively harmless to the hands and to metal instruments.

Adrenal histochemistry of some freshwater and marine teleosts

The adrenal histochemistry of seven freshwater and six marine species of teleosts was studied and correlated with thyroidal and gonadal activity. The adrenal tissue of each of the 92 fish utilized was subjected to each of the following histochemical reactions: lipids (Sudan black B), phospholipids, cholesterol, ascorbic acid, chromaffin reaction, iodate reaction, and appropriate controls. Adrenal, thyroid and gonadal tissues also were fixed in alcoholic Bouin's fluid and stained with hematoxylin and eosin or PAS (thyroid). The adrenocortical tissue of all species were positive for all the components normally reported in the vertebrate adrenal cortex. However, the marine and sexually mature freshwater species yielded more intense reactions. This was correlated with greater thyroid activity and is considered an indication of a more active secretory state. The possible effects of gonadal activity are discussed. Further, the significance of the marked histochemical variation at the cell level within an individual is also discussed. The chromaffin cells of all species except one yielded positive results with the chromaffin reactions and the iodate reaction. It was not possible to differentiate epinephrine- from norepinephrine-secreting cells because of the considerable interspecific variation in response and the scattered nature of the chromaffin cells.

Adrenal histochemistry of some freshwater and marine teleosts

The adrenal histochemistry of seven freshwater and six marine species of teleosts was studied and correlated with thyroidal and gonadal activity. The adrenal tissue of each of the 92 fish utilized was subjected to each of the following histochemical reactions: lipids (Sudan black B), phospholipids, cholesterol, ascorbic acid, chromaffin reaction, iodate reaction, and appropriate controls. Adrenal, thyroid and gonadal tissues also were fixed in alcoholic Bouin's fluid and stained with hematoxylin and eosin or PAS (thyroid). The adrenocortical tissue of all species were positive for all the components normally reported in the vertebrate adrenal cortex. However, the marine and sexually mature freshwater species yielded more intense reactions. This was correlated with greater thyroid activity and is considered an indication of a more active secretory state. The possible effects of gonadal activity are discussed. Further, the significance of the marked histochemical variation at the cell level within an individual is also discussed. The chromaffin cells of all species except one yielded positive results with the chromaffin reactions and the iodate reaction. It was not possible to differentiate epinephrine- from norepinephrine-secreting cells because of the considerable interspecific variation in response and the scattered nature of the chromaffin cells.

The staining of nervous elements by the Bodian method I. The influence of factors preceding impregnation

ABSTRACT The stainability of neural elements is determined during the various stages of tissue preparation which precede impregnation. The hypothesis (Romanes, 1950) that variations in the physiological state of the neurones at the time of fixation might influence the stain was examined. Feeding and anaesthesia did not influence the staining of gastric nerve fibres under the conditions of the experiments. A study was made of fixing fluids for Bodian staining. A picric-acetic-alcohol mixture and alcoholic Bouin were convenient and effective, each being superior to formaldehyde-acetic-alcohol. For overnight fixation formaldehyde solution neutralized with calcium carbonate was satisfactory, but where prolonged fixation is permissible formaldehyde neutralized with excess of magnesium carbonate gave superior results. Attention was drawn to the probability that the final stain could be improved by ‘pretreatment’ of mounted sections before silvering. Dewaxed sections were treated individually with 27 substances from a variety of chemical groups to determine the effects on subsequent Bodian staining. Nitric and sulphuric acids, formaldehyde solution neutralized with magnesium carbonate, methyl alcohol, and perhaps sodium carbonate contribute in different ways towards better preparations. In particular pretreatment with nitric acid suppresses the background stain, especially in smooth muscle; sulphuric acid increases the number of fine nerve fibres displayed, and methyl alcohol facilitates the staining (in formaldehyde-fixed tissues) of nerve fibres that are otherwise difficult to demonstrate, such as those in the renal cortex, which are almost certainly post-ganglionic sympathetic fibres.

A Redescription and Life-History of Gigantocotyle explanatum (Creplin, 1847) Nasmark, 1937 (Trematoda: Paramphistomidae) from India

Gigantocotyle explanatum (Creplin, 1847) Nasmark, 1937 is a very common parasite of the domestic ruminants in India and is found in the liver, bile ducts and gall bladder, usually accompanying infection with Fasciola. In the adult stage the parasites are non-pathogenic, or only slightly so, but immature forms when present in large numbers cause amphistomiasis which in the majority of the cases proves fatal to the host. This amphistome has been reported from Bos taurus indicus and Bubalis bubalis from India, Burma, Ceylon, Indo-China and the Philippines (Nasmark, 1937; Dawes, 1946). Except for a short and incomplete account given by Srivastava (1944) and a study of the structure of the miracidium by Ozaki (1951), no account of the life history of Gigantocotyle explanatum is available. Since considerable difficulty was found in the identification of the parasite and there still seems to be some confusion, a short diagnosis of Gigantocotyle explanatum is included here for the sake of convenience and future reference. The redescription is based on a large number of specimens studied as whole mounts and serial sections, collected from buffalo at Lucknow and with which the present work was carried out. As regards the synonymy of the species, reference is made to Fischoeder (1903, 1904), Maplestone (1923), Travassos (1934), Dawes (1936) and Nasmark (1937). For the purposes of the present work Gigantocotyle bathycotyle (Fischoeder, 1901) Nasmark, 1937 is considered a synonym of Gigantocotyle explanatum. MATERIALS AND METHODS The adult amphistomes were obtained from buffalo slaughtered locally and a large number of gravid specimens were always available throughout the year. The gravid specimens were identified and the eggs obtained by teasing the uterus in a small amount of distilled water. When a large number of eggs had thus been collected from several specimens the eggs were freed from debris by decantation and kept in covered petri dishes. During the summer months the water of the dishes was changed twice daily but only once during the winter months. The eggs were kept at laboratory temperature during the summer months and in a constant temperature cabinet at 82? F ? 2 F during winter months. A large amount of material was examined for the study of all the larval stages. Distilled water and 0.3% salt solution were used as mounting media. Usually the larval stages were examined in living condition without staining but the following intra vitam stains were also found useful: Neutral red, brilliant cresyl blue, methylene blue, methyl green and Bismarck brown. For making permanent preparations the material was fixed in warm alcoholic Bouin's fluid or Susa's fluid and stained with Ehrlich's haematoxylin, borax carmine, acetic acid alum carmine and Semichon's carmine. For sectioning, the material was fixed in alcoholic Bouin's fluid, cleared in cedarwood oil, embedded in wax and cut at 6-12 microns. The sections were stained with Ehrlich's haematoxylin and eosin, Heidenhain's iron haematoxylin and Mallory's triple stain. For the study of the cercaria, the ordinary methods gave poor results. Serum of sheep was used as a mountant for cercariae for studying the excretory system. For the study of the genital rudiments Schneider's acetocarmine was found to be an ideal stain and was used as described by Singh (1955). The permanent preparations were, however, not satisfactory.