Light and transmission electron microscopic studies of the third stage larvae of anisakis simplex.

Objective: The purpose of this study was to demonstrate the structures of the third stage larva of Anisakis simplex in marine fish by using light microscope (LM) and transmission and transmission electron microscope (TEM). Methods: L3 Anisakis simplex were processed for conventional light and electron microscopic studies. Results: The body wall of the L3 A. simplex is composed of three layers, the cuticle, hypodermis and somatic muscular layer from the superficial to deep surface of the worm, respectively. The cuticle is thick, functions as a protective barrier and barring the antigenic molecules. Furthermore, TEM reveals that the cuticle is subdivided into cortical, middle and basal layers from outer to inner part of the cuticle. This layer is highly filamentous and the filaments arrange randomly in several directions. The hypodermis is a thin layer which functions as the cuticular productive layer. The lateral hypodermal cords are bilobed. The somatic muscular layer is composed of a single row of muscle cells, which lie along the long axis of the body. The gastrointestinal tract of the worm is a straight tube, lined with stratified epithelium and surrounded by the basal lamina. The intestinal epithelial cells contain various organelles, which its luminal surface presents numerous microvilli for absorption of nutrient molecules. Conclusion: The cross section of LM and TEM can be used to distinguish the nematode species, as the LM reveals the lateral cords of L3 A. simplex are bilobed structures which are different from other species.

Light and electron microscopic studies of the structural damages of acanthamoeba cysts by pouzolzia indica methanolic extract fraction 4 solution.

Objective : This study was to investigate the morphological changes and ultrastructural damages of the Acanthamoeba cysts after treated with the minimal cysticydal concentration (MCC) of Pouzolzia indica methanolic extract fraction 4 solution by light and electron microscopies. Materials and Methods : Acanthamoeba cyst which prepared followed the method of Roongruangchai K15,16 were adjusted to the final concentration of 104 cysts/ml and treated with 1:4 soltion of fraction 4 Pouzolzia indica methanolic extract, the Thai medicinal plant. which was the minimal cysticidal concentration (MCC) 16. The cysts then, were centrifuged and the pellets were prepared for light, scanning and transmission electron microscopies. Result : Light micrographs showed the cytoplasmic clumping and some empty cyst wall. Transmission electron microscopy showed the ruptures of both ectocyst and the endocyst wall with some opercula damages. The cytoplasm aggregated and clumped. Scanning electron microscopy show steps of damage which started from shrinkage and collapsed of the cyst walls, then the cysts began to bulge and swell resulted in decreasing in the wrinkles of the cyst walls. The cytoplasmic contents drain out from the cyst wall and finally the cyst walls were ripped and torn into small pieces. Conclusion : Pouzolzia indica methanolic extract fraction 4 at the MCC of 1:4 caused structural damages to the Acanthamoeba cyst including shrinking of the cyst walls with the ruptured of the ectocyst, endocyst and operculum, edema and break out into pieces. The active ingredients of this Thai medicinal plant should be further studied as this can eventually be one of the regimen for the treatment of the Acanthamoeba keratitis or as a solution for cleaning the contact lens or contact lens case solution.

Phagocytosis: Mechanism of uptaking the nutrition of the embryonic cells.

Objective : To demonstrate the mechanisms of the embryonic cells, the epiblast, the mesoblast and the hypoblast of the chick embryo uptaking their nutrition by transmission electron microscopic (TEM) point of view. Materials and Methods : The chick embryos of about 18-27 hours incubation were used, the primitive streak and the 7 somites stages of the embryo were processed for routinely transmission electron microscopic study at the region of primitive streak, where the mesoblast originates from the epiblast. These stages correspond to the 15 days and 20 days of the human embryos, respectively. Results : The ultrastructural features of the epiblast, mesoblast and the hypoblast were observed. The epiblast cells arrange themselves as stratified columnar epithelium. The apical cells are columnar while the deeper cells are bottle-shaped and the deepest cells are round and separated to become the mesoblast. The superficial surfaces of the apical cells show many pseudopodia some of which curve to join with the adjacent. The clear materials are completely surrounded when the encompassing plasma membranes fuse and the membrane surround the engulfed material forms a vacuole, known as the phagosome, which detaches from the plasma membrane to float freely within the cytoplasm. The phagosome is then in some way recognized by one or more lysosomes which fuse with the phagosome to form the secondary lysosome. When the digestion is completed, the lysosomal membrane ruptures, discharging its contents into the cytoplasm. Undigested material remains within the membrane-bound vesicles called residual bodies, the contents of which later discharge at the cell surface by exocytosis. These processes also occur at the external surfaces of the hypoblast cells which arrange themselves as the simple squamous epithelium. The ventral plasma membrane shows many pseudopodia and also the series of phagocytotic process. The numerous clear vacuoles distribute in the cytoplasm of the epiblast and the hypoblast. These vacuoles are the nutritional uptake of the cell from the outside environment, the epiblast gets its nutrition from the albumin while the hypoblast from the egg yolk. Concluison: TEM evident shows clearly that numerous clear and unclear vacuoles distribute throughout the cytoplasm of the epiblast and hypoblast. These vacuoles are the phagosomes of several stages of phagocytosis, and serve as the nutritions for the cells, as the cells are still young and also have no definitive organelles to produce their own materials. This findings also give another excellent model of explaining the series of the phagocytotic process.

The origin of endoderm: Transmission electron microscopic point of view.

Objective: To show the transmission electron microscopic (TEM) evidence to confirm that the endoderm originates from the epiblast of the primitive streak or from other sources. Methods: 60 fertilized Leghorn hen’s eggs were used in this study by incubating the eggs for about 18-27 hours at 38oC, then the chick embryos of the primitive streak stage to 7-somite stage were further processed for routine TEM study at the region of the primitive streak. Results: The epiblast proliferates and accumulates to form the primitive streak at the midcaudal of the embryonic disc from 18-27 hours incubation which corresponds with the early third week of the human embryo. TEM evidence shows that the epiblast at the primitive streak is the stratified columnar type of epithelium while the hypoblast is the simple squamous and the mesoderm cells are irregular in shape. The process of gastrulation begins with the formation of the filopodia of the epiblast by numerous protrusions of the plasma membrane from lateral side of the cell. These structures initiate the separation of the contacted cells. The deepest epiblast cells separate first while the superficial epiblast cells exhibit the desmosome between the adjacent cells. The separated epiblast cells are bottle-shaped with numerous filopodia and gradually change the shape into round or oval cells which migrate in the space between the epiblast and hypoblast. Some of these migrate to the hypoblast and contact with the hypoblast, the mesoblasts lose the filopodia and gain more close contact to the hypoblasts which become a very thin sheet of cells. The facing cell membrane later gradually disappears and the mesoblast then occupies the region of pre-existing hypoblast. There is no evidence that the mesoblast displaces the pre-existing hypoblast laterally to form the extraembryonic endoderm. Conclusion: These are TEM evidences that the epiblast of the primitive streak separates and migrates to form the mesoblast and some contact with the hypoblast. The later process appeared to reveal that the mesoblast compresses the hypoblast until the facing plasma membrane disappears and occupies the region of the pre-existing hypoblast.

Several fractions of pouzolzia indica methanolic extract were lethal to the acanthamoeba cyst: in vitro study.

Objective: The purpose of this study was to compare the least concentrations of 5 fractions of Pouzolzia indica methanolic extract which can be lethal to the cyst form of the Acanthamoeba spp. Methods: Acanthamoeba spp. was isolated from a keratitis patient and was cultured using nonnutrient agar plates enriched with heat-killed E.coli for seven days at room temperature for the production of mature cysts. The cysts were harvested, washed in normal saline solution and adjusted to the final concentration of 104 cysts/ml. They were mixed with several dilutions of each fraction of Pouzolzia indica methanolic extract. After incubation for 1 hour, they were washed and centrifuged to remove the herbal extract supernatants. The cysts were recultured in the same medium for 7 days to confirm that they were all dead. Results: Pouzolzia indica methanolic extract fraction No. 1 which was eluted by water could not kill the cyst, while the crude extract (Fraction C) could at the concentration of 1:2. The fraction No.2 which was eluted by water: methanol had the minimal cysticidal concentration of 1:4, fraction No.3 which was eluted by methanol had the minimal cysticidal concentration of 1:8 and the fraction No.4 which was eluted by ethyl acetone had the minimal cysticidal concentration of 1:4. Concluison: Our results demonstrated that the Pouzolzia indica methanolic extract of several fractions can be cysticidal to an Acanthamoeba cyst, and this can modified to be a better disinfecting solution for contact lens cases.

Povidone – iodine induced changes in the cyst of Acanthamoeba spp.: transmission electron microscopic study.

Objective: This study was to investigate morphological changes and fine structures of the cyst form of Acanthamoeba after treatment with various concentrations of povidone – iodine (BetadineTM) using transmission electron microscopy. Methods: Acanthamoeba spp. were isolated from patients with amebic karatitis and obtained from the cultures on 3% non-nutrient agar (NNA) plates seeded with heat killed Escherichia coli (NNA-E coli) with incubation at 30°C for 7 days. The cysts were harvested and washed in ameba saline solution and adjusted to a final concentration of 104 cysts/ml. Various concentrations of povidone-iodine were put in the microtiter plate wells. The minimum cysticidal concentration was the lowest concentration that there was no excystment after 1 week of incubation. The cysts were prepared for routine transmission electron microscopy to determine the structural and organelle damages. Results: Structural damages were observed in the cysts treated with povidone-iodine of 0.04% dilution. Many cysts showed shrinkage of amoeba from the cyst wall and there was a slight withdrawal of the cytoplasm from the cyst wall. Many cysts were ruptured and broken into small pieces. Conclusion: Structural damages were observed in the cysts treated with 0.04% dilution of povidone-iodine solution or more than that. The damage started with pores produced in the cyst wall and the loss of water, shrinkage and loss of the cytoplasm of the inside cell from the cyst wall, followed by breaking of the cyst wall and the inside cell into small pieces.

Comparing lethal dose of povidone-iodine and virkon® to the acanthamoeba cyst: In vitro study.

Objective: To compare the least concentration of Povidone-iodine and Virkon®, phenolic-based compound with accelerated hydrogen peroxide and potassium peroxymonosulfate, which can cause a lethal effect to the cyst form of the Acanthamoeba spp. Methods: Acanthamoeba spp. was isolated from the keratitis patient and was cultured using amonoxenic medium supplement with heat killed Escherichia coli and incubated for up to seven days for the production of mature cysts. Acanthamoeba cysts on the culture plates were mixed with several dilutions of Povidone-iodine to compare to several dilutions of Virkon®. After incubation for 1 hour, they were washed and centrifuged to remove the chemical supernatant. The pellets of the mature cyst were viewed by a light microscope for seven days, and further recultured on the monoxenic medium to confirm that the cysts were all died (cysticidal) for seven days. Results: Povidone-iodine 0.04% and Virkon® 0.25% solutions were the least concentrations which could cause a lethal effect to the Acanthamoeba cyst. Conclusion: Our results demonstrated that both the Povidone-iodine and Virkon® showed antiamoebic activity. The further study should be done to determine whether Povidone-iodine and Virkon® can be used as a disinfecting solution for contact len cases.

Pollen grains in stool examination may lead to misdiagnosis for the parasite ova: 3 case reports.

Three cancer patients were admited in Siriraj Hospital for chemotherapy. Routinely, the blood, the urine and stool examination have to be done for checking up / excluding other diseases. In the stool examinations of these patients, light microscopically revealed abundant oval-shaped organism-like structures. They looked like the ova stage of several parasites which could not give the diagnosis by the light microscopic level. Therefore the ultrastructural examination was performed by scanning and transmission microscopes. The TEM revealed clearly that they were not of animal structure because their walls were made up of plant tissue - cellulose. They were concluded to be the pollen grains of plants by comparing to the figure and description from textbooks of palynology, the study of pollen grains. They may be the pollen grains of the herbal medicine which were consumed by the cancer patients as an alternative treatment of the cancer. If light microscopic level cannot give the exact diagnosis, electron microscopy may be helpful.

The ultrastructure of the gravid uterus of Brugia pahangi, another rich source of antigen of the filarial parasite.

The gravid uterus with zygotes and microfilariae in utero of Brugia pahangi, a rich source of antigen as revealed by a recent immunofluorescent technique, were studied ultrastructurally. The epithelial cells of uterus show ultrastructural features of synthetically active cells. Their secretions may provide nutrients for the egg in utero. On the basal side, the uterine epithelial cells may also secrete substances to form the basal lamina of the uterus which is rather thick and irregularly fused with the basal lamina lining the body wall where the pseudocoelomic cavity is obliterated. For the most part, the uterine basal lamina contains uniform granular material of moderate electron density. There are also elongated visceral muscle cells embeded in it, and which surround the uterus, with adjacent cells overlapping. The gravid uterus contains several stages of developing microfilariae within its lumen, the cleaving zygotes are also present at another level. The morula of zygotes are composed of several closely packed cells surrounded loosely by their own egg shell membranes. The egg shell becomes more convoluted as development proceeds. The egg shell surrounding the developing microfilariae in utero is secreted by the uterine epithelium. This structure later becomes the sheath of circulating microfilariae, and is highly antigenic as indicated by intense labeling with fluorescent antibodies.

The possible antigenic sources of brugia pahangi.

The antigenic sources of adult and the third larval (L3) stages of Brugia pahangi were detected by indirect immunofluorescent technique. Six panels of antisera were used, including human antisera against Brugia malayi and Wuchereria bancrofti, cat antisera against B.malayi and B.pahangi and jird antisera against B.malayi and B.pahangi as primary antibodies. All antisera gave the same results, although four of the six were not infected by B.pahangi. This indicates non-species specificity, and B.pahangi, B.malayi and W.bancrofti must share most of the common antigenic molecules. All antisera reacted well with the surface of L3 B.pahangi in the whole mount preparation. This indicates non-stage specificity. The most intense fluorescence was located at the epicuticle, the basal lamina lining the body wall, the gut and the reproductive tract, the egg shell in utero and the sperm. The hypodermis, the muscle cells, the cuticle beneath the epicuticle, the epithelial cells of the gut and the reproductive tract showed moderate fluorescence. The least fluorescence was observed in the egg interior.