Plasma etching and ashing: a technique for demonstrating internal structures of helminths using scanning electron microscopy.

Plasma etching and ashing for demonstrating the three-dimensional ultrastructure of the internal organs of helminths is described. Adult worms of the cestode Caryophyllaeides fennica were dehydrated through an ethanol series, critical point dried (Polaron E3000) and sputter coated with 60% gold-palladium (Polaron E5100) and glued to a standard scanning electron microscope (SEM) stub positioned as required for ashing. After initial SEM viewing of worm surfaces for orientation, stubs were placed individually in the reactor chamber of a PT7150 plasma etching and ashing machine. Worms were exposed to a radio frequency (RF) potential in a low pressure (0.2 mbar) oxygen atmosphere at room temperature. The oxidation process was controlled by varying the times of exposure to the RF potential between 2 to 30 min, depending on the depth of surface tissue to be removed to expose target organs or tissues. After each exposure the oxidized layer was blown from the surface with compressed air, the specimen sputter-coated, and viewed by SEM. The procedure was repeated as necessary, to progressively expose successive layers. Fine details of organs, cells within, and cell contents were revealed. Ashing has the advantage of providing three dimensional images of the arrangement of organs that are impossible to visualize by any other procedure, for example facilitating testes counts in cestodes. Both freshly-fixed and long-term stored helminths can be ashed. Ashing times to obtain the desired results were determined by trial so that some duplicate material was needed.

Larval trypanorhynchs (Platyhelminthes: Eucestoda: Trypanorhyncha) from black-scabbard fish, Aphanopus carbo and oceanic horse mackerel, Trachurus picturatus in Madeira (Portugal).

Four different types of trypanorhynch postlarvae were found attached to the stomach mucosa, external stomach wall or free in the body cavity of two marine fishes from Madeira, namely blackscabbard fish, Aphanopus carbo and oceanic horse mackerel, Trachurus picturatus. Morphological features shown by SEM indicated that the postlarvae belonged to the species Tentacularia coryphaenae, Sphyriocephalus tergestinus, Nybelinia lingualis and possibly N. yamaguitii. Prevalence [mean intensity (range)] of T. coryphaenae, S. tergestinus and Nybelinia spp. in A. carbo (n = 135) was 12.6% [1.65 +/- 1.27(1-6)], 5.9% [1.57 +/- 0.79 (1-3)] and 2.2% [1.33 +/- 0.58 (1-2)] respectively. The prevalence of T. coryphaenae and S. tergestinus showed some seasonality, with a rise in prevalence of T. coryphaenae corresponding to a decrease in prevalence of S. tergestinus. However these differences were not significant. In T. picturatus (n = 304) only N. lingualis was found at a prevalence of 9.6%. Both S. tergestinus and N. lingualis were recovered only from the stomach mucosa or external stomach wall, while T. coryphaenae was observed either attached to the stomach mucosa or free in the visceral cavity of the fish. The paper presents the first scanning electron micrographs (SEM) of Sphyriocephalus tergestinus and a new geographical record of N. lingualis in T. picturatus.

Cystacanths of Bolbosoma vasculosum in the black scabbard fish Aphanopus carbo, oceanic horse mackerel Trachurus picturatus and common dolphin Delphinus delphis from Madeira, Portugal.

Cystacanths of the acanthocephalan, Bolbosoma vasculosum Rudolphi 1819, were found to be encapsulated in the connective tissues of the viscera of the black scabbard fish, Aphanopus carbo and oceanic horse mackerel, Trachurus picturatusfrom Madeira, Atlantic Ocean. Juvenile worms were obtained from the intestine of a stranded common dolphin, Delphinus delphis, also from Madeira. Cystacanths were 11-15 mm long, with a proboscis of 18-19 longitudinal rows, eight hooks per row, and two sets of trunk spines. Overall, the morphology and dimensions of the proboscis, neck and trunk corresponded to previous descriptions. Scanning electron microscopy of the proboscis structures and trunk spines is provided for the first time. The prevalence of B. vasculosum in A. carbo increased with fish length. There were no statistical differences in the prevalence and intensity of infection between seasons. The intensity of infection was similar for male and female fishes, but there were significant differences in relation to length, longer fishes having heavier infections. Aphanopus carbo from Madeira represents a new host record and a new geographic location for B. vasculosum.

Freeze fixation-dehydration as a method of preparation of Gyrodactylus (Monogenea) for SEM.

Freeze fixation-dehydration was used for the first time in the preparation of attached Gyrodactylus for SEM viewing. The technique provided instant immobilization of specimens before death and negligible shrinkage throughout the fixation-dehydration process. Comparisons of sample means of two linear measurements of attached opisthaptors showed 20% more shrinkage of Gyrodactylus fixed using 10% neutral buffered formalin than those which were freeze fixed. Freeze fixation-dehydration was excellent for the study of gross external morphology of attached Gyrodactylus. However, the freeze fixation-dehydration process may cause disruption of intracellular structural components making delicate tissues brittle and more prone to damage during subsequent manipulation.

Soft X-ray contact imaging of nucleolar chromatin using synchrotron radiation: a comparative scanning and transmission electron microscope study.

Contact images (CI) of dehydrated, nucleolar chromatin from amphibian oocytes have been produced by soft X-rays from a synchrotron radiation source. These CI have been compared with the morphology of the original chromatin as seen in scanning and transmission electron microscopes. The quality and informational content of the CI depend very much on certain preparative procedures. The following factors have a marked effect on image quality and need to be carefully controlled: the total X-ray dose, the time and nature of development and the distance of the specimen from the photoresist. The preparation of the chromatin itself, providing that it is critically point dried, is less important. By following a regime of high X-ray dose, sufficient for penetration of the rather thick chromatin rings, and gentle development so that fine detail is not dissolved from the resist surface, it has been possible to obtain images which closely resemble the original chromatin, although the detailed resolution of the CI is not as clear. The smallest biological structures clearly resolved in the CI are ribonucleoprotein granules, which vary in size from 200 to 800 nm. However, by further refinement of preparative conditions it should be possible to improve on the informational content of these images.

Effects of calcium antagonists and calcium-buffered salines on wound healing in Xenopus early embryos.

The role of calcium in the process of wound closure in Xenopus early embryos was studied. Embryos were wounded in the presence of the calcium antagonists D-600 and TMB-8 or in calcium-buffered salines, and the effects on wound healing were observed by scanning electron microscopy. D-600 and TMB-8 inhibit wound closure and these antagonists appear to act synergistically since their combined effect is greater than their individual effects. Experiments with calcium-buffered salines suggest that wound closure can proceed in the presence of low extracellular calcium. In all conditions there is a correlation between the degree of wound closure and the shapes of the cells at the wound margin; closing wounds are accompanied by cells elongated radial to the wound, gaping (non-closing) wounds are accompanied by cells stretched tangential to the wound. Thus the results suggest that calcium influx may not be a requirement for the changes in cell shape which accompany, and probably effect, wound closure in Xenopus early embryos.