Millonig's Buffer Research Articles

Induced arthritis, hepatocytes cytotoxicity and non-steroidal anti-inflammatory drugs (NSAIDs) – experimental study

This study aimed to histologically evaluate the pharmacological activity of meloxicam, COX-2 inhibitor, on liver function in arthritic rats. Meloxicam is a non-steroidal anti-inflammatory drug (NSAID), COX-2 inhibitor, generally prescribed in the area of rheumatology. Thus, 18 Wistar rats were divided into 3 groups with 6 animals each: G1 (Negative Control), G2 (Arthritis induced with Zymosan [Zy]), G3 (Arthritis induced with Zymosan [Zy] and treated with meloxicam). The G3 treatment started on the 21st day after induction. The animals were sacrificed, by overdose of Ketamine/Xylazine, seven, 14 and 21 days after the beginning of the meloxicam treatment. Liver samples were collected from the animals, fixed in Millonig buffer containing 10% formaldehyde and processed for conventional histological analysis. G1 animals showed absence of inflammation and degeneration of hepatocytes. In the G2 group, a slight cytoplasmic alteration was observed at seven and 14 days. At 21 days, intense cytoplasmic and nuclear disorganization was detected, accompanied by cell necrosis. In G3, large spacing between sinusoid capillaries was detected. In some 14-day-old animals, amyloidosis was observed, with the presence of a large number of vacuolated hepatocytes involved in an amorphous mass of necrotic cells. Most of the hepatocyte nuclei had compacted chromatin, evidencing cellular degeneration. Meloxican, used as an anti-inflammatory in the synovial region, showed that, in addition to its beneficial effects for the treatment of rheumatological diseases, it is capable of, when administered orally, promoting a high level of liver damage.

Lack of Tau Proteins Rescues Neuronal Cell Death and Decreases Amyloidogenic Processing of APP in APP/PS1 Mice

Lack of tau expression has been reported to protect against excitotoxicity and to prevent memory deficits in mice expressing mutant amyloid precursor protein (APP) identified in familial Alzheimer disease. In APP mice, mutant presenilin 1 (PS1) enhances generation of Aβ42 and inhibits cell survival pathways. It is unknown whether the deficient phenotype induced by concomitant expression of mutant PS1 is rescued by absence of tau. In this study, we have analyzed the effect of tau deletion in mice expressing mutant APP and PS1. Although APP/PS1/tau(+/+) mice had a reduced survival, developed spatial memory deficits at 6 months and motor impairments at 12 months, these deficits were rescued in APP/PS1/tau(-/-) mice. Neuronal loss and synaptic loss in APP/PS1/tau(+/+) mice were rescued in the APP/PS1/tau(-/-) mice. The amyloid plaque burden was decreased by roughly 50% in the cortex and the spinal cord of the APP/PS1/tau(-/-) mice. The levels of soluble and insoluble Aβ40 and Aβ42, and the Aβ42/Aβ40 ratio were reduced in APP/PS1/tau(-/-) mice. Levels of phosphorylated APP, of β-C-terminal fragments (CTFs), and of β-secretase 1 (BACE1) were also reduced, suggesting that β-secretase cleavage of APP was reduced in APP/PS1/tau(-/-) mice. Our results indicate that tau deletion had a protective effect against amyloid induced toxicity even in the presence of mutant PS1 and reduced the production of Aβ.

Accelerated Human Mutant Tau Aggregation by Knocking Out Murine Tau in a Transgenic Mouse Model

Many models of human tauopathies have been generated in mice by expression of a human mutant tau with maintained expression of mouse endogenous tau. Because murine tau might interfere with the toxic effects of human mutant tau, we generated a model in which a pathogenic human tau protein is expressed in the absence of wild-type tau protein, with the aim of facilitating the study of the pathogenic role of the mutant tau and to reproduce more faithfully a human tauopathy. The Tg30 line is a tau transgenic mouse model overexpressing human 1N4R double-mutant tau (P301S and G272V) that develops Alzheimer's disease-like neurofibrillary tangles in an age-dependent manner. By crossing Tg30 mice with mice invalidated for their endogenous tau gene, we obtained Tg30xtau(-/-) mice that express only exogenous human double-mutant 1N4R tau. Although Tg30xtau(-/-) mice express less tau protein compared with Tg30, they exhibit signs of decreased survival, increased proportion of sarkosyl-insoluble tau in the brain and in the spinal cord, increased number of Gallyas-positive neurofibrillary tangles in the hippocampus, increased number of inclusions in the spinal cord, and a more severe motor phenotype. Deletion of murine tau accelerated tau aggregation during aging of this mutant tau transgenic model, suggesting that murine tau could interfere with the development of tau pathology in transgenic models of human tauopathies.

A light and transmission electron microscope study of the distribution and ultrastructural features of peripheral nerve processes in the extra-retinal layers of the zebrafish eye

The distribution and ultrastructural features of peripheral nerve processes in the extra-retinal layers of the eyes of the zebrafish, Danio rerio (Hamilton), were investigated using light and transmission electron microscopy. A comparative study of the quality of preservation provided by three different fixation procedures revealed no consistently striking general differences. However, somewhat subjectively, the fixative containing Millonig's buffer did consistently provide better fixation of myelin. Overall, nerve processes, depending on the site studied, were distributed as either (1) bundles (in the choroid near the optic nerve head and in the choroid adjacent to the limbus), (2) linear arrays (in the junction between the sclera and cartilage and in the choroid adjacent to the retina) or (3) individual units (in the choroid under the cartilage or in the sclera). Both myelinated and unmyelinated processes were identified in these locations. Myelinated processes usually contained both neurofilaments and neurotubules, but a few apparently contained only neurofilaments. Unmyelinated processes usually contained mainly neurotubules, but a few apparently contained only neurofilaments. Taken together, these findings indicate innervation of extra-retinal structures, as seen in zebrafish, is highly conserved among vertebrates, further supporting the use of zebrafish as a model for the vertebrate visual system.

Whole‐specimen histopathology: a method to produce whole‐mount breast serial sections for 3‐D digital histopathology imaging

To develop a method for preparing diagnostic-quality, whole-mount serial sections of breast specimens while preserving 3-D conformation. This required supporting the fresh specimen prior to breadloafing and refining the conventional tissue processing method. The overall goal is to use digital images of whole-specimen histopathology to improve the estimation of extent of disease. To maintain a 3-D conformation, the specimen is suspended in 3.5% agar at 55 degrees C. The block is sliced at 5-mm intervals. Sectioning is performed after extended fixation in 4% formaldehyde from paraformaldehyde in 0.1 m Millonig's buffer, followed by paraffin processing using a non-routine schedule and extended paraffin infiltration. Whole-mount serial breast sections are produced with features of equal or superior quality to that which can be achieved using conventional methods. The method is compatible with some immunohistochemical stains but requires further optimization for others. The technique is currently suitable for research applications. With the reduction in processing time achievable with microwave-assisted processing, there is the potential for its use as a routine clinical method. This tool may improve the accuracy of margin estimates and identification of multifocality in breast cancer; further evaluation is necessary.

Alzheimer's Disease-Like Tau Neuropathology Leads to Memory Deficits and Loss of Functional Synapses in a Novel Mutated Tau Transgenic Mouse without Any Motor Deficits

Tau transgenic mice are valuable models to investigate the role of tau protein in Alzheimer's disease and other tauopathies. However, motor dysfunction and dystonic posture interfering with behavioral testing are the most common undesirable effects of tau transgenic mice. Therefore, we have generated a novel mouse model (THY-Tau22) that expresses human 4-repeat tau mutated at sites G272V and P301S under a Thy1.2-promotor, displaying tau pathology in the absence of any motor dysfunction. THY-Tau22 shows hyperphosphorylation of tau on several Alzheimer's disease-relevant tau epitopes (AT8, AT100, AT180, AT270, 12E8, tau-pSer396, and AP422), neurofibrillary tangle-like inclusions (Gallyas and MC1-positive) with rare ghost tangles and PHF-like filaments, as well as mild astrogliosis. These mice also display deficits in hippocampal synaptic transmission and impaired behavior characterized by increased anxiety, delayed learning from 3 months, and reduced spatial memory at 10 months. There are no signs of motor deficits or changes in motor activity at any age investigated. This mouse model therefore displays the main features of tau pathology and several of the pathophysiological disturbances observed during neurofibrillary degeneration. This model will serve as an experimental tool in future studies to investigate mechanisms underlying cognitive deficits during pathogenic tau aggregation.

Applications of Soft X-Ray and other Microscopy Techniques to Elucidate the Structure of Parasitic Metazoa

Abstract The small size of many parasitic organisms requires the use of election microscopy for adequate elucidation of their structure. While both transmission and scanning electron microscopy can provide complementary results which allow considerable degree of structural correlation, each technique has its inherent limitations. Since previous studies have demonstrated that soft X-ray microscopy could be used to study parasitic protozoa and provide new information, the objective of this study was to determine whether soft X-ray microscopy could also be used to elucidate the morphology of small metazoa to complement the data obtained by other microscopy techniques. Newborn larvae, approximately 7 μm × 110 μm in size, of parasitic nematode Trichinella spiralis were used as a model system. Some of the larvae, deposited by adult females maintained in vitro, were isolated and processed for examination by transmission and scanning electron microscopy as described in our previous studies, others were fixed in 4% glutaraldehyde (Millonig's buffer) and examined in the X-ray microscope XM-1, and in the BioRad MRC 1024 confocal laser (krypton/argon) microscope of the Advanced Light Source, Berkeley National Laboratory.

Effects of isoproterenol treatment on the parathyroid glands in golden hamsters of different ages, with special reference to the frequency of lipid droplets

Recently, we reported that there is an increase in lipid droplets in the parathyroid glands of isoproterenol-treated adult golden hamsters in which the synthesis and release of parathyroid hormone are stimulated. This investigation was undertaken to study the effects of different ages on lipid droplets in the parathyroid glands of the golden hamsters after administration of isoproterenol.The percent area occupied by lipid droplets in the parathyroid glands of 15, 20 and 25-day-old golden hamsters after 1 h of treatment with isoproterenol and the frequency of lipid droplets of 1-monthold (young), 3-month-old(adult) and 18-month-old(senile) animals after 0.5, 1 and 3 h of treatment with isoproterenol were investigated. The parathyroid glands of the control and isoproterenol-treated animals were removed and immersed in a mixture of 2.5% glutaraldehyde and 2% OsO4 in Millonig's buffer at pH7.4 for 1 h, dehydrated through increasing concentrations of acetone and embedded in Epon 812.

Effect of depolarization by potassium proprionate on synaptic vesicle diameter and distribution of the snake motor endplate

The morphological effects of elevated potassium (K+) at the neuromuscular junction of garter snake twitch muscle fibers were examined in vitro. Elevated K+ depolarizes the motor neuron terminals and causes quantal release of acetylcholine. It has been shown in amphibians that prolonged (30-60 min) depolarization with high K+ depletes terminals of synaptic vesicles ; but causes no change in the average diameter of the vesicles. In the snake, however, we have shown previously that miniature endplate currents (MEPCs) are present at high frequency even after 1-2 hours of exposure to high K+ . This indicates that in the snake, synaptic vesicles are not totally depleted by depolarization with high K+. In this study, we examined the effect of depolarization with high K+ on the distribution and diameter of synaptic vesicles.Intercostal muscle preparations were exposed for 15 minutes to Ringer's + solution containing either a control (1.2mM) or high (70mM) concentration of K+ Preparations were fixed immediately in 1% glutaraldehyde for 15 minutes and washed with Millonig's buffer. The whole mount muscle preparations were then fixed for 1 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2; rinsed three times in fresh buffer and post-fixed in 2% OsO4 in the same buffer for 30 min, rinsed in fresh buffer and dehydrated in a graded ethanol series to 100% and then transferred into a 2% solution of uranyl acetate for 20 min to en-block stain the whole mounts. The preps were then transferred back to 100% ethanol to complete the dehydration and embedded in a mixture of Embed 812/Araldite 502 (Hard) between two microscope slides that had been precoated with Formen- Trennmittel Liquid Release Agent and polymerized overnight at 70°C.The whole mounts were then removed from the microscope slides and examined on a compound light microscope where the neuromuscular junctions on the twitch muscle fibers could be identified, marked and cut out of the preps. These pieces of the whole mounts were then remounted into a slot on the end of a pre-cast Embed-Araldite block with a drop of fresh resin and polymerized.

Utilization of replicas to show gold-probe binding to sperm cells for SEM and TEM

The introduction of gold particles bound to various probes has facilitated studies of cell surface and intracellular tracing. One problem has been the inability to show surface binding of small gold particles without embedding and thin-sectioning the sample. Our work has shown that small gold probes of 10-15 nm diameter attached to lectins can be detected at surface sites by the use of replicas. The replica retains the three-dimensional aspect of the cell surface while fixing the gold-probe in place. Murine spermatazoa labelled with gold-Con A were replicated directly or replicated following a silver intensification. Results showed that the probes remained at their binding sites and were clearly visible at the ultrastructural level.Murine epididymal and caudal sperm were collected in PBS pH=7.4. Sperm were sedimented twice through PBS, then spread onto clean glass slides and fixed with 3% glutaraldehyde in Millonig's buffer.

The Occurrence of a Virus of the Pox Group in a Field Population of Chironomus salinarius Kieffer (Diptera: Chironomidae) in Italy

A larval population study on Chironomus salinarius Kieffer in two saltwater lakes surrounding the city of Orbetello, central Italy, was recently reported by Ali and Majori (1984). In this study, a part (18%) of the C. salinarius larval population in a sample taken from one station in June 1982 was patently infected with an entomopoxvirus. Further sampling of that station in June-July 1982 showed the overall disease incidence to be 6% (A. Ali, unpublished). A detailed description of the two lakes (East Lake and West Lake) is available in Ali and Majori (1984). The diseased larvae occurred at station 8 of sampling row G in the West Lake where the lake received domestic and industrial discharges. Patently infected larvae were recognized in the laboratory by the appearance of whitish areas beneath the integument of the entire body caused by massive accumulations of virus polyhedral bodies (PB). Infected 4th instars were randomly separated and placed in a rearing chamber (Biever 1965) to study their survival. Wet mount preparations (in saline) of larval smears of diseased larvae were observed under a phase-contrast microscope and the PB from cells of different host specimens were randomly measured. For electron microscopy, infected 4th instar C. salinarius were fixed in a 2.5% glutaraldehyde in Millonig buffer (pH 7.4) for 1 hour. After rinsing in the buffer, the tissues were postfixed for an hour in 1% OSO4, dehydrated in acetone series and embedded in Spurr's resin. Thin sections were examined with a Siemen's Elmiskop 102 and Zeiss EM 10 C electron microscope. Suspensions of purified virions (Huger et al. 1970) were negatively contrasted with 2% phosphotungstic acid at pH 7.2 and placed on carbon grids. Microscopic examination of smears of infected larvae revealed oval shaped PB with a mean size of 7.30 x 5.52 p.m but ranging in length from 3.4-13.23 pLm and width of 2.47-9.26 ,um. Phase-contrast microscopy showed that a variable number of PB may

Effects of Monoclonal IgG Antibodies on Eimeria tenella (Coccidia) Sporozoites

Although monoclonal antibodies have been generated against certain coccidia (Carosi et al., 1980, Boll. Inst. Sieroter Milan. 59: 25-30; Danforth, 1982, J. Parasitol. 68: 392-397; Sethi et al., 1980, J. Parasitol. 66:192-196), little is known about their effects on various stages of these parasites. In a previous study that used transmission electron microscopy (TEM), we found that 1A, 9D, and 3D3II monoclonal IgG antibodies caused complement-mediated lysis of Eimeria tenella sporozoites (Speer et al., 1983, J. Protozool. 30: 548-554). When viewed by scanning electron microscopy (SEM), we found that sporozoites of E. tenella exposed to these monoclonal antibodies without complement differed considerably in surface texture and size from normal sporozoites. Monoclonal antibodies were obtained as described previously (Kohler and Milstein, 1975, Nature 256: 495-497; Speer et al., 1983, loc. cit.). Sporozoites of E. tenella were incubated for 30 min at room temperature (22 C) with normal mouse ascites fluid or with 1A, 9D, or 3D3II monoclonal IgG antibodies (heat-inactivated) that had been diluted 1: 1 in a Tris-buffered glucose saline solution (pH 7.4) consisting of 10 mM Tris, 10 mM glucose, 170 mM NaCl, and 10% fetal calf serum. Sporozoites were then fixed in 2% glutaraldehyde in Millonig's phosphate buffer (pH 7.2). Sporozoites suspended in Millonig's buffer were captured on Nuclepore filters (0.6 Mm pore size) that had been coated previously with normal mouse serum in order to ensure adherence of the sporozoites. Glutaraldehyde fixative was then pulled through the filter to fix the sporozoites to the filter. Sporozoites on filters were rinsed in buffer, placed in 1% osmium tetroxide for 20 min, rinsed in buffer, dehydrated in ethanol, critical-point dried in a Samdri critical-point dryer, mounted on metal studs, coated with 25 nm of gold: palladium (60:40), and examined with a Zeiss Novascan 30 SEM. All measurements were made at a magnification of X7,000. Significant differences between dimensions of sporozoites exposed to normal ascites fluid and of those exposed to monoclonal antibody were determined by Student's t-test. By using indirect fluorescent antibody assay, lA, 9D, and 3D3II monoclonal IgG antibodies were found to have titers of 1:1.3 X 106, 1:1.2 X 104, and 1:1.6 X 106, respectively. Sporozoites exposed to normal ascites fluid measured 10.01 X 2.02 ,m (9.5-10.5 X 1.8-2.2 Mm; n = 20), whereas those exposed to 9D, 1A, or 3D3II were 9.36 X 2.02 ,um (8-10 X 1.8-2.3 Am; n = 20), 7.97 X 2.18 Am (7-9 X 2-2.5 inm; n = 20), and 6.8 X 2.39 ,m (6-8 X 2.2-2.6 ,im; n = 20), respectively. Sporozoites incubated with 1A and 3D3II were significantly shorter than those incubated with normal ascites fluid (P < 0.0005); 9D caused no significant difference in length (Figs. 1-4). Sporozoites exposed to 1 A and 9D did not differ significantly in width when compared to those exposed to normal ascites fluid, whereas those exposed to 3D3II were significantly wider (P < 0.05). Sporozoites incubated with normal ascites fluid or 9D had relatively smooth surfaces with small protrusions (Figs. 1, 2), whereas those exposed to 1A or 3D3II had rough or wrinkled surfaces with blebs (Figs. 3, 4). Although immune sera have been found to have an immobilizing or lytic effect on sporozoites and merozoites of E. tenella (Bums and Challey, 1965, J. Parasitol. 51: 660-668; Herlich, 1965, J. Parasitol. 51: 847-851), the role that immune sera plays in immunity to this parasite is still unknown. By using SEM, Witlock and Danforth (1982, J. Protozool. 29: 441-445) found that immune chicken serum caused surface bulges, a fibrinous coat and complement-me-

Dense granules: artefacts caused by fixation with glutaraldehyde, osmiumtetroxide and millonig buffer

Dense granules: artefacts caused by fixation with glutaraldehyde, osmiumtetroxide and millonig buffer

Visualization of the Platelet-Plasma Interface

Morphological and cytochemical studies of platelets have reported a surface coat, or glycocalyx, external to the plasma membrane (1). Biochemical analyses have likewise confirmed the highly adsorptive properties of platelets as transporters of coagulation factors (2). However, visualization of the platelet membrane by conventional EM procedures does not reflect this special relationship between the platelet and its plasma environment. By the routine method of alcohol-propylene oxide dehydration for Epon embedding, the lipid bilayer nature of the platelet membrane appears similar to other blood cells (Fig. 1). A new rapid embedding technique using dimethoxypropane (DMP) as dehydrating agent (13) has permitted ultrastructural analyses of the surface features of the platelet-plasma interface.Aliquots of human or rabbit platelet-rich plasma (PRP) were added to equal volumes of 6% glutaraldehyde in Millonig's buffer at 37° for 45 minutes, rinsed in buffer and postfixed in 1% osmium in Millonig's buffer for 45 minutes.

Electron Microscopic Observations on Virus-Like Particles of a Catfish Papilloma

Papillomas in catfishes have been described (1) but the presence of viruses in these tumors has not (2). This report is concerned with the study of a papilloma found on the lower lip of a brown bullhead (letalurus nebulosus) which had been frozen prior to arrival at our laboratory.Tumor tissues were thawed in 2.5% glutaraldehyde and postfixed in 1$ osmium tetroxide. Both fixatives were prepared in Millonig's buffer. Fixation, washing, dehydration, and Epon embedding were accomplished according to conventional electron microscopic techniques.Examination of thin sections revealed virus-like particles in epidermal cells which constituted the major part of the neoplasm (Fig. 1). No particles were found in the connective tissue surrounding the epidermal papillae. The cells containing particles were usually isolated from one another, had a spindle or fusiform shape, and exhibited many cytoplasmic extensions in random directions. Their nuclei were pleomorphic and displayed irregular nuclear envelopes with relatively large lacunae. Vesiculation of the cytoplasm was extensive, apparently caused by dilations of the endoplasmic reticulum.

Peculiar artefacts after fixation with glutaraldehyde and osmium tetroxide.

Peculiar electron dense granules were observed, in otherwise well preserved structures of the heart muscle and kidney of the rat after a 5-10 min perfusion, a 2 h fixation in the modified Karnovsky solution, followed by a postfixation in Millonig buffer containing 1% OsO4. The granules appeared most frequently on membranes, the surface of the red blood cells and in pinocytotic vacuoles, in stained and unstained sections alike. No artefacts were produced when the same buffer was used for both pre- and postfixation, or when the postfixative contained Veronal buffer.

A Golgi-electron microscope method for insect nervous tissue.

Golgi's light microscope method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 2 1/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50 mum sections are made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.

Fine Structural Classification of Chromophobe Adenomas of the Human Pituitary Gland

Chromophobe pituitary adenomas arise from adenohypophysial cells and fail to exhibit cytoplasmic staining with conventional acid or basic dyes by light microscopy. The aim of the present work was to study the electron microscopic features of these tumors, to separate them into distinct entities and to correlate their fine structural appearances with secretory activity.Among 48 surgically removed various pituitary adenomas 30 tumors were found which, based on the tinctorial characteristics of the cytoplasm, corresponded to chromophobe adenomas. For electron microscopic investigation pieces of these tumors were fixed in 2.5 per cent glutaraldehyde in Sorensen's buffer, post fixed in 1 per cent osmium tetroxide in Millonig's buffer, dehydrated in graded ethanol and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate.By electron microscopy it was possible to separate chromophobe adenomas into 3 distinct entities: 1) adenomas consisting of sparsely granulated growth hormone cells (7 cases).

Formation of Centrioles in Some Endocrine Adenomas

Centrioles, known to play a fundamental role in mitotic division, are regarded as representing self-replicating organelles. They occur with varying frequency in different normal cells and exhibit uniform appearances showing no signs indicative of duplication such as budding or division. In order to obtain a deeper insight into centriole formation and evolution, cases of parathyroid and pituitary adenomas with a high centriole population were investigated by electron microscopy.The material used for the present study consisted of 4 chief cell adenomas of the parathyroid gland, 7 sparsely granulated pituitary growth hormone cell adenomas and 1 mixed cell pituitary adenoma composed of sparsely granulated growth hormone cells and prolactin cells. These adenomas were selected from a large number of cases (26 parathyroid adenomas and 49 pituitary adenomas). All specimens were obtained by surgery from various patients with hyperparathyroidism or acromegaly. Small pieces of tissue were fixed immediately after removal in 2.5 per cent glutaraldehyde in Sorensen's buffer, postfixed in 1 per cent osmium tetroxide in Millonig's buffer, dehydrated in graded ethanol and embedded in Epon 812.

Fine Structure of the Schizonts and Merozoites of Eimeria acervulina in the Chicken

Fine structural changes observed during development of merozoites were similar in the 1st and later asexual generations of Eimeria acervulina in the chicken. Merozoites were formed at the surface of multinucleated schizonts. The anlage of the merozoite first appeared as a cone-shaped elevation of the surface, in which the inner membrane complex and the organelles of the apical complex developed. The nucleus, Golgi complex, and other organelles were incorporated into the developing merozoite while it was attached by the posterior end to the residual cytoplasm. The inner membrane of the pellicle terminated anteriorly at a polar ring and posteriorly at a thickening suggestive of a posterior polar ring. Twenty-two subpellicular microtubules extended along the merozoite from the anterior polar ring to the posterior end. The nucleus and its Golgi complex were situated in the posterior half of the merozoite and mitochondria, endoplasmic reticulum, refractile bodies, numerous micronemes, and 2 rhoptries in the anterior half. Mitochondria and endoplasmic reticulum were found posterior to the nucleus. The conoid had a spiral structure and 2 anterior preconoidal rings. A micropore just anterior to the nucleus was well developed in the lst-generation merozoites. Polysaccharide granules were found only in the 2ndand 3rd-generation merozoites. Entry of merozoites into villus epithelial cells caused deformity of microvilli. Secondand 3rd-generation schizonts lying close to the brush border deformed microvillar filaments in the superficial web zone. Developing schizonts were within parasitophorous vacuoles in host cells whose endoplasmic reticulum and numbers of mitochondria had increased. Mature schizonts were in cells whose organelles, including the nucleus, had degenerated. Tyzzer (1929) described the life cycle and the morphology of the various stages of Eimeria acervulina in the chicken. E. acervulina has since been frequently used to study the effect of intestinal coccidiosis on the chicken host (Preston-Mafham and Sykes, 1970; Turk and Stephens, 1967; Hein, 1968), but as yet these effects have not been examined at the fine structural level. Lee and Millard (1971a) described the fine structure and development of the macrogamete and oocysts of E. acervulina. The ultrastructure of the schizonts and merozoites and of the changes in host cells parasitized by schizonts are described in this paper. MATERIALS AND METHODS Two-week-old, male White Leghorn chicks, raised coccidia-free, were inoculated with sporulated oocysts of Eimeria acervulina as previously described (Fernando and McCraw, 1973). They were killed at 12, 24, 36, 48, and 56 hr, and 3, 4, and 5 days postinfection, and tissues were processed for electron microscopy. The birds killed up to 56 hr postinfection received 4 million sporulated oocysts and those killed at later periods Received for publication 2 March 1973. * Supported by Ontario Health Research Grant No. PR.297 and by the Ontario Ministry of Agriculture and Food. received 500,000 sporulated oocysts each. A section of the duodenum, about 2 inches proximal to the entry of the bile duct, was cut into pieces, 1 to 2 mm square, and fixed in 3% glutaraldehyde in Millonig's buffer at 4 C. The tissues were fixed from 4 to 48 hr, the best results often being obtained with longer fixation periods. They were washed for 24 hr or longer, first in Millonig's buffer and then in 0.2 mi sucrose solution, buffered at pH 7.4 with 0.1 M phosphate, postfixed in 1% osmium tetroxide in Millonig's buffer for 1 hr, washed in the same buffer, and dehydrated through a graded series of acetone, all at 4 C. The tissues were embedded in Epon and sectioned on a Reichert OM ultramicrotome. Ultrathin sections were mounted on 200-mesh copper grids and stained with lead citrate (Venable and Coggeshall, 1965), followed by a saturated solution of uranyl acetate in 50% ethanol. They were examined and photographed at 60 kv in a Phillips EM 300 electron microscope.