Retaining ionic concentrations during in vitro storage of tissue for microanalytical studies

When human or animal tissue is to be investigated by X-ray microanalysis, it is sometimes necessary to store the tissue between removal from the organism and freezing. However, when excised tissue is stored in buffer, the elemental concentrations in the cell may change. In the present study, it was attempted to develop a storage buffer that would retain the cellular elemental concentrations close to their in situ values. To start, the NaCl component in Krebs–Ringer buffer was exchanged for K-gluconate and KCl for NaCl. This buffer was called a ‘100% high K⁺ solution’. Starting from this solution, part of the K-gluconate was replaced by an equivalent amount of NaCl. Incubation of excised rat liver (4 °C, 4 h) in 85% high K⁺ solution resulted in retention of cellular Na, K, Ca, S and Mg concentration most closely to the in situ state, whereas cellular Cl was retained best when the tissue was incubated in 75% high K⁺ solution. For rat submandibular gland, incubation in 80% high K⁺ solution resulted in optimal retention of cellular Na, K, Ca, P, S and Mg, while Cl was retained best in a 70% high K⁺ solution. Based on these results, an optimally modified Krebs–Ringer solution for the liver would consist of 119 mM K⁺, 26 mM Na⁺ and 45 mM Cl^?. An optimally modified Krebs–Ringer bicarbonate solution for the submandibular gland would be composed of 96 mM K⁺, 53 mM Na⁺ and 46 mM Cl^?. After incubation in the modified solutions (at 4 °C), cellular Na, Mg, S, Cl, K and Ca in both tissues were maintained close to the in situ state throughout a 6-h incubation. The cellular P concentration was reduced after incubation for 1 h; thereafter, in the liver cells it remained at this lower level for the rest of the incubation, whereas in the submandibular gland tissue it increased again after 4 h. The increase in cell volume (oedema) was less in tissue stored in the modified solutions, than in the 100% high K⁺ solutions. Incubation in high Na⁺ buffers (4 °C, 6 h) resulted in a progressive increase in the percentage of cells showing trypan blue uptake. A similar increase in trypan blue uptake was seen in the modified solution, but this increase levelled off after 4 h. After cholinergic stimulation in high Na⁺ solution (25 °C, 1 min), the expected decrease in cellular Cl concentration was seen in submandibular gland cells that had previously been preserved (4 °C, 4 h) in the modified solution, but not in those that had been preserved in the 100% high K⁺ solution.     

Elemental concentrations in isolated rat thymocytes prepared for cryofixation in the presence of different media

Rat thymocytes were isolated in suspension and the effect of preparing the cells for cryofixation in the presence of different media on the elemental content was investigated using the technique of X-ray microanalysis. Cells prepared in the different media showed variation in the concentrations of Na, K and Cl. The isolated cells were incubated at 310 K for 1 h to allow recovery from isolation. There was a decrease in Na and Cl content after incubation. The thymocyte population was disturbed by suspension in medium containing dextran, and this resulted in a number of cells with high concentrations of Na and low concentrations of K. These cells did not take up vital dye. Thymocytes were also prepared for freezing by using high-speed centrifugation to concentrate the cells. Thymocytes prepared by this method showed values for concentrations of Na, K and Cl similar to published values for these cells using other methods of estimation. There were, however, consistent differences in Na content between the cells prepared in Hanks' balanced salt solution and those prepared in serum. Factors which affect the apparent concentration of Na and Cl in isolated cells are discussed.     

Confocal laser scanning microscopic analysis of ectopic sublingual gland‐like tissue inside the hamster submandibular gland

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3′‐diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO‐positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous‐like cells were found in resin‐embedded semi‐thin sections of the DAB‐reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance‐mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity‐negative secretory granules and seromucous cells with PO activity‐positive spot‐like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron‐dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron‐dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland‐like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct. Microsc. Res. Tech. 76:1284–1291, 2013. © 2013 Wiley Periodicals, Inc.     

Application of elemental microanalysis to elucidate the role of spherites in the digestive gland of the helicid snail Chilostoma lefeburiana

In this case study we present an application of different analytical electron microscopic methods in biology, to elucidate their usefulness in such investigations. Using analytical electron microscopy, spherites in the digestive gland cells of the helicid snail Chilostoma lefeburiana were examined at three stages: just before the non-feeding period of over-wintering in November, in the middle of over-wintering in February and at its end in March. A detailed characterization of changes in the elemental composition of the spherites was characterized by a combination of transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDXS), electron energy-loss spectroscopy (EELS) and energy filtering TEM (EFTEM). During over-wintering, the spherites passed the following changes. Before over-wintering in November, they consisted of striking concentric layers of electron-dense and electron-lucent zones, while in February and March they showed clear empty zones between materials of different electron density. In November spherites, C, O, Ca, P, Cl, Fe, Si, Na, K, Mg and S were detected, whereas in February ones C, O, N, Cl, Si and S were found and only C, O, N, Si and Cl were detected in March spherites. It is suggested that the elements missing in February and March were used in different physiological processes during over-wintering, like (1) the maintenance of the appropriate elemental composition of the internal environment, (2) accumulation of non-toxic waste materials that cannot be metabolized and (3) avoiding potential intoxication by contamination with toxic metals.     

Low-temperature X-ray microanalysis of the differentiating vascular tissue in root tips of Lemna minor L

The fracture faces of bulk-frozen tissue offer a number of advantages for the analysis of diffusible elements. They are easy to prepare, remain uncontaminated, and, unlike most frozen-hydrated sections, can be shown to exist in a fully hydrated state throughout examination and analysis. Root tips of Lemna minor briefly treated with a polymeric cryoprotectant are quench frozen in melting nitrogen. Fractures are prepared using the AMRAY Biochamber, lightly etched if necessary to reveal surface detail and carbon coated while maintaining the specimen at 110 K. The frozen-hydrated fracture faces are analysed at 110 K using the P/B ratio method which is less sensitive to changes in surface geometry and variations in beam current. The method has been used to investigate the distribution of seven elements (Na+, Mg⁺⁺, P, S, Cl^?, K+ and Ca⁺⁺) in the developing vascular tissue of the root tip. The microprobe can measure relative elemental ratios at the cellular level and the results from this present study reveal important variations in different parts of the root. The younger, more actively dividing cells, appear to have a slightly higher concentration of diffusible ions in comparison to the somewhat older tissues which have begun to differentiate into what are presumed to be functional vascular elements.     

Methods for X-ray microanalysis of epidermis: the effect of local anaesthesia

The effect of local anaesthesia on the elemental content of cells in human epidermis was studied by electron probe X-ray microanalysis. Local anaesthesia with lidocaine was given by intracutaneous injection within 1 min prior to taking a skin biopsy. Biopsies taken without local anaesthesia were used as controls. Lidocaine with or without adrenaline caused a significant increase in the concentrations of Na and Cl, and a decrease in the concentration of K in the cells of the stratum basale and the stratum spinosum, compared with the control samples. The presence of adrenalin in the anaesthetic did not change the effect of lidocaine. The effects of local anaesthesia have to be considered in planning and interpretation of clinical applications of X-ray microanalysis.     

Imaging intracellular elemental distribution and ion fluxes in cultured cells using ion microscopy: a freeze-fracture methodology

A freeze-fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze-drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the ～0·5 μm lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K-low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze-fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo-sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK₂ rat kangaroo and L5 rat myoblast cultures.     

The intracellular distribution of ions and water in rat liver and heart muscle

Local dry mass concentrations of intracellular compartments in rat heart muscle and liver cells were estimated by quantitative electron microscopy and X-ray microanalysis of ultrathin frozen-dried cryosections. The results were used to calculate elemental concentrations per litre of compartment water from the X-ray microanalytical data. Water fractions were between 80.3 ± 1.3% of wet weight in the decondensed chromatin and only 45.1 ± 1.7% in mitochondria of liver cells. The lowest water fraction in heart muscle cells was also found in mitochondria. The ionic concentrations found in the cytoplasm of liver cells and in the myofibrils are in accord with the electroneutrality rule and in osmotic equilibrium with the extracellular concentrations. The concentrations of Na, K, Cl and P both in the cytoplasm and in the regions of decondensed chromatin within the nuclei were found to be equal. However, in regions of condensed chromatin K⁺ concentrations were found to be much higher than expected for a Donnan distribution of ions free in solution. Most probably the activity coefficient for K⁺ is lower in the condensed chromatin than in the decondensed or in the cytoplasm. The same holds true for the A-band as compared to the I-band in heart muscle cells. A sequestration of K⁺ was measured also in the rough endoplasmic reticulum (RER) of hepatocytes. The Cl^? concentration in mitochondria both in heart muscle and liver cells has been measured far in excess of what might be expected from a Nernstian distribution. A coupled inward Cl^? transport in mitochondria must, therefore, be assumed.     

Quick sampling and perpendicular cryosectioning of cell monolayers for the X-ray microanalysis of diffusible elements

A quick sampling and preparation method for freezing of cell monolayers is described. The cells are grown on a large Formvar film supported by a frame of polystyrene. A polyvinylpyrrolidone (PVP) solution is applied to one side of the film forming a flat disc when frozen with a pair of pliers precooled in liquid nitrogen. The PVP solution provides the specimen with sufficient strength and may be used as an elemental standard for absolute quantification if salts of known concentrations are added. Manipulation of the cells prior to freezing is thus restricted to a minimum, which eliminates possible harmful treatments like scraping and centrifugation. The procedure is quickly performed, the freezing being completed within 30 s of the cells having been removed from the culture well. The analytical results reveal low and stable Na: K ratios. Our results confirm that cells in vitro are comparable to cells in vivo with respect to elemental composition.     

Electron and ion imaging of gland cells using the FIB/SEM system

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga⁺ ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga⁺ ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.     

Intramandibular glands in different castes of leaf‐cutting Ant,Atta laevigata (Fr. Smith, 1858) (Formicidae: Attini)

Intramandibular glands have been poorly studied in polymorphic ants, where the differences between castes were unsufficiently scrutinized. Leaf‐cutting ants possess one of the most complex systems of communication and labor division, which is polymorphic well as age polyethism, and makes them an ideal model for the study of intramandibular glands. This study has investigated the occurrence of intramandibular glands in female castes and subcastes of Atta laevigata. The mandibles of the queen, medium, and minor workers, and soldiers were submitted to histological, histochemical, ultrastructural, and morphometric analyses. The class‐3 gland cells and the epidermal gland with a reservoir were found in all the castes. The queens and soldiers showed a higher number of class‐3 gland cells, distributed within the mandible as well as a greater gland size in comparison to the workers. The histochemical tests, periodic acid‐Schiff (PAS), mercury‐bromophenol, and Nile blue, were similar for the class‐3 gland cells and epidermal glands with a reservoir. However, the tests evidenced differences between the castes, with carbohydrates strongly positive in all of them, whereas neutral lipids were found in the queen and soldiers. The protein was weakly positive in the queen, whereas in the soldier, medium, and minor workers these reactions were strongly positive in the intramandibular glands. Our findings in A. laevigata suggest that intramandibular glands are directly involved in labor division and consequently in chemical communication between the castes. Microsc. Res. Tech. 78:603–612, 2015. © 2015 Wiley Periodicals, Inc.     

Morphological and elemental integrity of freeze-fractured,freeze-dried cultured cells during ion microscopic analysis

The effects of progressive ion beam bombardment on freeze-fractured, freeze-dried cultured cells during ion microscopic (SIMS) analysis were studied with scanning electron microscopy (SEM) and ion microscopy. The freeze-fracture, freeze-dry sample preparation method was generally found to preserve cell morphology to a level far exceeding the spatial resolution of the ion microscope, with splitting at the nuclear envelope being the most commonly observed artefact. SEM monitoring of surface topography of an NRK-49F fibroblast after various ion bombardment doses showed relatively uniform erosion of cellular material, with some apparent selective retention of small cytoplasmic granules. Prolonged bombardment produced no detectable lateral elemental translocation. ⁴¹K+/²⁴Mg+ signal ratios from Swiss 3T3 fibroblasts and RBL rat basophilic leukaemia cells were shown to vary generally by less than 10% during the course of extended ion bombardment. GM0415 human skin fibroblasts containing engorged lysosomes characteristic of Hurler's Syndrome were used to evaluate the effects of ion bombardment during a typical analysis session, where ion images of ³⁹K+, ²³Na+, ⁴⁰Ca+ and ²⁴Mg+ are sequentially recorded. This cell line was chosen as a worst-case system, because these cells are often thinly spread and possess extreme surface topography. Thin cell edges were shown sometimes to sputter away during analysis, giving misleadingly low ion signals from these regions in some ²⁴Mg+ micrographs. Various nonuniform sputtering phenomena occurring in the submicrometre spatial domain had little or no measurable impact on local intensities in ion micrographs, indicating that freeze-dried, freeze-fractured cells are sampled in a sufficiently uniform fashion that quantitative ion microscopic evaluations of intracellular elemental levels in the general cytoplasmic or nuclear regions are feasible.     

Rapid freezing,cryosectioning, and x-ray microanalysis on cardiac muscle preparations in defined functional states

Electrically stimulated heart muscle preparations can be quickly frozen in undercooled propane at defined times of the mechanically controlled contraction cycle. The apparatus for triggered freezing of the muscle strips in undercooled propane is described in detail. Freeze substitution of some strips after freezing shows the degree of ice crystal formation without the potential interference of artifacts introduced later by cryosectioning and freeze drying. Ultrathin longitudinal and transversal cryosections are cut with a LKB cryoultramicrotome at temperatures of −130 to −140°C, freeze-dried at 10⁻⁶ Torr vacuum and carbon-coated before analysis. The freeze-dried cryosections are analyzed in a Siemens Elmiskop 102 electron microscope equipped with a Kevex energy dispersive system, and the elemental concentrations (in mMol/kg d.w.) of Na, Mg, P, S, Cl, K, and Ca are determined in subcellular compartments of muscle frozen in different functional states. The methodology of quantitation, i.e, determination of elemental net peak and continuum, correction of continuum, preparation of standards, and deconvolution of overlapping peaks are described. The minimum detectable elemental concentration using the reported methods is in the range of a few mMol/kg d.w. This also applies to Ca, which can be accumulated in heart muscle in readily detectable amounts in intracellularly located stores as well as structures connected with the cell membrane. The present report shows that cryotechniques and x-ray microanalysis can be successfully applied to heart physiology.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

Optimal preparatory procedures of cryofixation for immunocytochemistry

To examine the optimal preparatory procedures of cryofixation for immunocytochemistry, the labeling density over the antigenic sites in cells processed by various protocols of freeze-substitution and embedding was quantitatively evaluated. Fresh tissue blocks of gerbil parotid gland were quickly frozen by a metal contact method using liquid helium and freezesubstituted with one of the following media: 4% OsO₄ in acetone or 0.4% OsO₄ in acetone or 0.3% glutaraldehyde in acetone. They were then embedded in either an Epon-Araldite mixture or Araldite 6005, which were polymerized at 60°C and 50°C, respectively. Some frozen samples substituted with aldehyde-containing acetone were embedded in Lowicryl K4M (polymerized at —30°C). Immunocytochemical localization of amylase was examined by indirect immunostaining by using antigerbil parotid amylase antibody and protein A/gold complex. Thin sections of epoxyresin-embedded materials were treated with oxidizing agents before immunostaining. The central dense core of heterogeneous secretory granules in the acinar cells was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The labeling density on thin sections of all the cryofixed materials examined was about 1.5 times or more as high as in those processed by conventional chemical fixation. The highest value of the labeling density was obtained from material which was substituted with 0.3% glutaraldehyde in acetone and embedded in Araldite 6005. Substitution with osmium-containing acetone appeared not to seriously affect immunoreactivity of the antigenic sites and was advantageous because of the distinctive images of membranes. Advantages and disadvantages of the individual protocols employed are discussed.     

Quasicrystalline Materials

SIMS matrix effects (mass interferences, sputter yield variations and practical ion yield variations) were evaluated in freeze-fractured, freeze-dried cultured cells at the ～0.5 μm spatial resolution of the Cameca IMS-3f ion microscope. Cell lines studied include normal rat kidney (NRK), 3T3 mouse fibroblast, L6 rat myoblast, chinese hamster ovary (CHO) and rat kangaroo kidney (PtK2) cells. High mass resolution studies indicated that the secondary ion signals of H^—, C^—, O^—, Na⁺, Mg⁺, CN^—, P^—, S^—, Cl^—, K⁺ and Ca⁺ were free from major mass interferences. However, a large mass interference was observed for nitrogen at mass 14. No significant sputtering yield difference between the nuclear and cytoplasmic compartments of the cells studied was observed. The subcellular distributions of the major (H, C, N and O) and minor (P, S, K, Cl, Na, Mg and Ca) matrix elements were found to be largely homogeneous with the exception of Ca, which was observed mainly in the cell cytoplasm. Practical ion yield variations were compared by three different approaches: (i) by the use of cells doped with known electrolyte concentrations, (ii) by quantitative ion implantation, and (iii) by analysis of the same cell with both electron probe and ion microscope. Each approach indicated an absence of significant practical ion yield differences between the nuclear and cytoplasmic regions of these specimens. These observations indicate that secondary ion signals in this type of sample are not significantly affected by local matrix effect variations. Hence, qualitative imaging of such specimens provides a true representation of subcellular elemental distribtions. These observations should allow the development of quantitative ion imaging methodologies and enhance the applicability of ion microscopy to biomedical problems.     

In vitro and in situ experimental model for X-ray microanalysis of intestinal epithelium

Intestinal chloride (Cl) transport is disturbed in a number of diseases. X-ray microanalysis can be used to study the distribution of Cl and other ions in intestinal epithelial cells. In this study it was attempted to establish an experimental system that retains the in vivo elemental composition of intestinal epithelial cells. An in vitro system was set up in which a segment of rat intestine was mounted in a bath and perfused with different fluids. The chloride in the bath or in the perfusion fluid could be exchanged for gluconate or bromide to determine the direction of chloride fluxes. An in situ system was set up in which the animal was anesthetized and a segment of the intestine was perfused with different solutions. In the in vitro experiments the concentration of Na and Cl in the epithelial cells increased and that of K decreased. These changes occurred within the first 30 minutes of incubation. Uptake of chloride occurred mainly from the bath, as seen in experiments where bromide was used as a chloride analog. The concentration gradient between bath and tissue determined the extent of chloride uptake. Addition of glucose to the perfusion fluid and bath did not improve the results. In the in situ system, preservation of the intracellular ion composition was better. Acceptable results were obtained with perfusion with Krebs-Ringer's buffer without glucose for 30 minutes. In this case, the elemental content of the cell did not change appreciably during incubation. If glucose was added, the Na concentration increased in comparison to the control, both in crypt and villus cells. It is concluded that the intestinal epithelium is a sensitive system, very prone to disturbance of its homeostasis. However, the in situ system can be used in studies of agonist-induced ion transport.     

Light and transmission electron microscopic structure of skin glands and dermal scales of a caecilian amphibian Gegeneophis ramaswamii,with a note on antimicrobial property of skin gland secretion

Amphibian skin secretions contain a variety of bioactive compounds that are involved in diverse roles such as communication, homeostasis, defence against predators, pathogens, and so on. Especially, the caecilian amphibians possess numerous cutaneous glands that produce the secretory material, which facilitate survival in their harsh subterranean environment. Inspite of the fact that India has a fairly abundant distribution of caecilian amphibians, there has hardly been any study on their skin and its secretion. Herein, we describe, using light microscopy and electron microscopy, two types of dermal glands, mucous and granular, in Gegeneophis ramaswamii. The mucous glands are filled with mucous materials. The mucous‐producing cells are located near the periphery. The granular glands are surrounded by myoepithelial cells. A large number of granules of different sizes are present in the lumen of the granular gland. The granule‐producing cells are present near the myoepithelial lining of the gland. There are small flat disk‐like dermal scales in pockets in the transverse ridges of the posterior region of the body. Each pocket contains 1–4 scales of various sizes. Scanning electron microscopic (SEM) study of the skin surface showed numerous funnel‐shaped glandular openings. The antibacterial activity of the skin secretions was revealed in the test against Escherichia coli, Klebsiella pneumoniae, and Aeromonas hydrophila, all gram‐negative bacteria. SEM analyses confirm the membrane damage in bacterial cells on exposure to skin secretions of G. ramaswamii.     

Structural and histological observations on the male reproductive system of adult red poplar leaf beetle Chrysomela populi Linnaeus, 1758 (Coleoptera: Chrysomelidae)

This study described the anatomy and histology of the male reproductive system in Chrysomela populi, which is an economically important species belonging to the family Chrysomelidae. Therefore, reproductive biology has been studied to combat this insect. As well as, the characters associated with the reproductive tract have been potential to discuss aspects of the system and to better understand the reproductive dynamics. The male reproductive system of C. populi has a pair of testes, a pair of vas efferentia and deferentia, a pair of seminal vesicles, a pair of accessory glands, an ejaculatory bulb, an ejaculatory duct, and an aedeagus. The testis consists of two flower-shaped lobes. Each testis has 20 sperm tubules (testicular follicles) containing cysts of germ cells at various developmental stages within the light orange peritoneal sheath. Testicular follicles are composed of three different (growth, maturation, and differentiation) zones. In the middle region of each testis joins with the vas efferens. The testis is attached to the seminal vesicle by a small stalk like vas efferens. In the lumen of the vas efferens, seminal vesicle, and vas deferens, sperms form clumps in the form of thin threads. The proximal end of the vas deferens is connected to the common ejaculatory duct. It joins with the ejaculatory bulb. Around the ejaculator bulb, there is a pair of convoluted, flat-surface tubular structure accessory glands. Posterior ejaculatory duct joins with the aedeagus.     

The morphology of the pineal gland of the yellow‐toothed cavy (Galea Spixii Wagler, 1831) and red‐rumped agouti (Dasyprocta leporina linnaeus, 1758)

The pineal gland is an endocrine gland found in all mammals. This article describes the morphology of this important gland in two species of Caviideae, namely the yellow‐toothed cavy and the red‐rumped agouti. Ten adult animals of the two species used in current analysis were retrieved from the Center for the Multiplication of Wild Animals (CEMAS/UFERSA) and euthanized. The glands were removed and photographed in situ and ex situ. They were fixed in a paraformaldehyde solution 4% or glutaraldehyde 2.5% solution and submitted to routine histological techniques respectively for light and scanning electron microscopy. Macroscopically, the pineal gland with its elongated structure may be found between the cerebral hemispheres facing the rostral colliculi. Microscopically, pinealocytes and some glia cells were predominant. Contrastingly, to the cavy's pineal gland, a capsule covered the organ in the agouti, with the emission of incomplete septa to the interior, which divided it into two lobules. Light and scanning electron microscopes failed to show calcareous concretions in the pineal gland. Based on the topography of the cavy's and agouti's pineal gland, it may be classified as supra‐callosum and ABC type. Microsc. Res. Tech. 78:660–666, 2015. © 2015 Wiley Periodicals, Inc.