Scanning electron microscopical comparisons of insect virus occlusion bodies prepared by several techniques

Samples of baculoviruses, a cytoplasmic polyhedrosis virus, and an entomopoxvirus were prepared by several techniques for study in the scanning electron microscope. The techniques which gave satisfactory definition of surface ultrastructure were: (1) double fixation in glutaraldehyde and osmium tetroxide with critical point drying; and (2) double fixation as in (1) with a further postfixation by ligand-mediated osmium binding using thiocarbohydrazide as the ligand (OTO method) followed by critical point drying. The latter technique was superior to the former technique. Air drying of samples gave acceptable but inferior definition when compared with critical point-dried samples prepared by these techniques.     

Apical surface topography of invaginating and noninvaginating cells. A scanning-transmission study of amphibian neurulae

A comparative study on scanning and transmission electron microscopy of apical ultrastructure in epithelia of the axolotl neurula (Ambystoma mexicanum, Cope) is presented. The aim of the work is to determine whether apical surface topography is correlated to other morphological features of the cells, and whether there are any ultrastructural differences between surfaces of invaginating and noninvaginating regions. Scanning specimens are prepared by critical point drying and benzene freeze-drying. Comparisons show that the scanning specimens are comparable to the standard transmission specimens with regard to surface topography.Apical surfaces are sculptured by folds and microvilli-like processes. Assessment of the relative abundance of surface projections shows that these occur in largest numbers on invaginating, bottle-shaped cells in the neural plate and in the notochord rudiment of stage-16 larvae. It is proposed that the surface projections may support apical narrowing in these cells, by facilitating endocytosis, and that they may be lateral attachment organelles. It is suggested that the morphogenesis of the invaginating cells may be the result of coordination between microfilaments, apical endocytosis, microtubules, and “adhesive peripheral surface projections.”     

Ultrastructural localization of the Pasteurella multocida toxin in a toxin-producing strain

Toxigenic strains of Pasteurella multocida produce the 147 kDa protein Pasteurella multocida toxin (PMT) which is responsible for the osteoclastic bone resorption in progressive atrophic rhinitis in pigs and induces such resorption in all experimental animals tested so far. In the present study we have carried out immunocytochemistry on formaldehyde- and glutaraldehyde-fixed ultracryocut P. multocida using a pool of monoclonal antibodies against different epitopes on PMT as the first layer and affinity purified rabbit anti-mouse IgG as the second layer. Goat anti-rabbit IgG conjugated with 5 nm gold particles was used as marker. The gold particles were silver-enhanced prior to examination in the transmission electron microscope. Whole bacteria were also immunostained after fixation and critical point drying and examined by scanning transmission electron microscopy. The results showed that PMT was located in the cytoplasm of P. multocida. PMT could not be detected on intact, undamaged P. multocida by scanning electron microscopy. Neither pili nor flagella could be detected on the surface of the negatively stained P. multocida strains investigated. PMT has a series of characteristics encompassed in the definition of an exotoxin. However, that PMT was not secreted by living intact P. multocida is unexpected for an exotoxin.     

A technique for quick fixation of whole Drosophila and other small insects for scanning electron microscopy

A procedure for fixing small insects in natural postures for scanning electron microscopy is reported. Anesthetized insects are partially restrained using a depression slide and a coverslip while preliminary fixation is carried out and wings and legs are positioned with a fine brush. Following this, fixation is completed and the insect is prepared for scanning electron microscopy by essentially standard procedures, which may include critical point drying. Figures illustrate, however, that critical point drying is not necessary for more rigid parts of the exoskeleton. Use of this procedure assures naturally disposed parts even when only a single specimen is available.     

A Technique for Quick Fixation of Whole Drosophila and Other Small Insects for Scanning Electron Microscopy

A procedure for fixing small insects in natural postures for scanning electron microscopy is reported. Anesthetized insects are partially restrained using a depression slide and a coverslip while preliminary fixation is carried out and wings and legs are positioned with a fine brush. Following this, fixation is completed and the insect is prepared for scanning electron microscopy by essentially standard procedures, which may include critical point drying. Figures illustrate, however, that critical point drying is not necessary for more rigid parts of the exoskeleton. Use of this procedure assures naturally disposed parts even when only a single specimen is available.     

Freeze-drying from tertiary butanol in the preparation of endocardium for scanning electron microscopy.

The scanning electron microscope appearances and shrinkage of blocks of canine endocardium prepared by freeze-drying directly, by freeze-drying after replacing tissue water with tertiary butanol (2-methyl propan-2-ol) and by critical point drying were compared. All three methods demonstrated endothelial cells which showed nuclear prominences, microvilli and intercellular boundaries. The microvilli varied in size and number from dog to dog but were generally less well defined in specimens freeze-dried from water. Shrinkage due to t-butanol dehydration was significantly less than that which occurred in ethanol in the critical point drying method. Overall the reduction in surface area was significantly less in specimens freeze-dried directly at -65 C (6.8%) than in those dried from t-butanol at -20 C (15.4%) and those prepared bly critical point drying (22.1%). However the amount of shrinkage observed in t-butanol treated tissue was not significantly different from that which was critical point dried. It was not possible to distinguish between comparable samples prepared by these two methods on the basis of their scanning electron microscopic appearances. Thus the relative simplicity and convenience of the t-butanol method, together with its saving of time, its use of standard freeze-drying equipment and the avoidance of ice-crystal artefact justify its consideration as an alternative method of preparing wet biological tissue for scanning electron microscopy.     

Freeze-Drying from Tertiary Butanol in the Preparation of Endocardium for Scanning Electron Microscopy

The scanning electron microscope appearances and shrinkage of blocks of canine endocardium prepared by freeze-drying directly, by freeze-drying after replacing tissue water with tertiary butanol (2-methyl propan-2-01) and by critical point drying were compared. All three methods demonstrated endothelial cells which showed nuclear prominences, microvilli and interoellular boundaries. The microvilli varied in six and number from dog to dog hut were generally less well defined in specimens freeze-dried from water. Shrinkage due to t-butanol dehydration was significantly less than that which occurred in ethanol in the critical point drying method. Overall the reduction in surface area was significantly less in specimens freeze-dried directly at -65 C (6.8%) than in those dried from t-butanol at -20 C (15.4%) and those prepared by critical point drying (22.1%). However the amount of shrinkage observed in t-butanol treated tissue was not significantly different from that which was critical point dried. It was not possible to distinguish between comparable samples prepared by these two methods on the basis of their scanning electron microscopic appearances. Thus the relative simplicity and convenience of the t-butanol method, together with its saving of time, its use of standard freeze-drying equipment and the avoidance of ice-crystal artefact justify its consideration as an alternative method of preparing wet biological tissue for scanning electron microscopy.     

An alternative SEM drying method using hexamethyldisilazane (HMDS) for microbial cell attachment studies on sub-bituminous coal

The use of hexamethyldisilazane (HMDS) as a drying agent was investigated in the specimen preparation for scanning electron microscopy (SEM) imaging of bacterial surface colonization on sub-bituminous coal. The ability of microbes to biofragment, ferment and generate methane from coal has sparked interest in the initial attachment and colonization of coal surfaces. HMDS represents an attractive alternative to critical point drying (CPD) in the imaging of cells on coal, negating the need for expensive equipment. Coal is easily fragmented into sub-micron particles, which can be problematic in critical point drying procedures. In this study, both individual and aggregated cells appeared well shaped with minimal occurrence of flattened cells, signifying the suitability of HMDS in cell attachment studies on sub-bituminous coal. In the absence of glucose, microcolonies of short and long cells showed similar positive results using this method. EPS shrinkage found in microcolonies was inevitable, though this enabled observation of points of attachment between cells and with coal, which would be less effective if the EPS was intact. Overall the use of HMDS drying is preferred over the more commonly used CPD method as it is safer, cheaper and more practical.     

Effect of kaolinite on the specific infectivity of reovirus

Abstract The infectivity of enteric viruses (e.g., poliovirus, rotavirus, reovirus) is prolonged when these viruses are adsorbed on naturally occurring particulates (sediments, clay minerals) in terrestrial and aquatic environments. Furthermore, in vitro assays of these and other particulate-associated viruses often display infectivity levels (specific infectivity) greater than those of the same concentration of viruses in the absence of particulates. This investigations attempted to identify interactions at the particulate-virus-cell interface and to define the mechanism(s) whereby the apparent infectivity of viruses is enhanced when complexed with particulates. Reovirus type 3 and the clay mineral, kaolinite, were used as the model systems. Scanning electron micrographs after critical point drying showed that kaolinite was not present on the surface of cell monolayers of L-929 mouse fibroblasts 3 h after inoculation with a kaolinite-reovirus complex. However, the virus was observed on the surface of the cells. No change in dispersion of the virus particles was observed nor was the integrity of the cell surface altered by kaolinite. These results indicated that kaolinite enhanced the transport of viral particles, in conjunction with diffusion and Brownian movement, to receptors for the reovirus on the cell surface.     

Scanning electron microscopy of cultured cells of Aedes aegypti

Cultured cells of Aedes aegypti were fixed with glutaraldehyde and prepared for scanning electron microscopy by four procedures: air drying, lyophilization, ethanol dehydration and air drying, and ethanol dehydration and critical point drying. Comparison of the resulting electron micrographs with phase contrast photomicrographs of living cells revealed that although cultured insect cells dried by the critical point method are not completely without artifacts, this method of preservation is superior to other techniques currently used.     

A New Method Using Hexamethyldisilazane for Preparation of Soft Insect Tissues for Scanning Electron Microscopy

A new rapid procedure for preparing soft internal tissues from insects that allows air drying was found to compare favorably with tissues prepared by critical point drying. In the new procedure, tissues were fixed in 1% glutaraldehyde, dehydrated through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes, and air dried. Tissues prepared by both the HMDS treatment and by critical point drying were coated with gold for scanning electron microscopy. Tissues prepared by the HMDS treatment did not shrink or distort upon air drying and excellent surface detail was preserved. The HMDS treatment required about 5 minutes, whereas the critical point drying procedure required about 1.5 hours.     

A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron microscopy

A new rapid procedure for preparing soft internal tissues from insects that allows air drying was found to compare favorably with tissues prepared by critical point drying. In the new procedure, tissues were fixed in 1% glutaraldehyde, dehydrated through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes, and air dried. Tissues prepared by both the HMDS treatment and by critical point drying were coated with gold for scanning electron microscopy. Tissues prepared by the HMDS treatment did not shrink or distort upon air drying and excellent surface detail was preserved. The HMDS treatment required about 5 minutes, whereas the critical point drying procedure required about 1.5 hours.     

Application of freeze-drying intact cells to studies of murine oncornavirus morphogenesis.

Using a method for freeze-drying intact cells, uninfected and murine leukemia virus (MuLV)-infected JLSV9 cell surfaces, as well as murine mammary tumor virus (MuMTV)-infected cell surfaces, were examined by electron microscopy. The 10-nm knobs of MuLV and the 5-nm spikes of MuMTV were clearly revealed on the surfaces of budding viruses and were also found dispersed over the cell surface. The MuLV knobs are randomly arranged on the virus surface, whereas the MuMTV spikes are much more ordered. Because freeze-fractured budding viral envelopes are devoid of intramembranous particles, the observed surface particles do not appear to be merely accentuated intramembranous particles. This technique should permit further analysis of the morphogenesis of viral envelopes without the need for externally applied labels.     

Intercellular attachments between calcified collagenous tissue forming cells in the rat

Summary Osteoblasts of the young rat cranium, and cementoblasts and odontoblasts of young rat molars were prepared by ethanol freeze-fracture prior to critical point drying for scanning electron microscopy (SEM) as well as conventional transmission electron microscopy (TEM) techniques. Critical point drying causes shrinkage which separates the lateral intercellular contacts between neighbours in the same sheet in the case of cementoblasts and osteoblasts, but not those between odontoblasts. These differences are considered to be of functional significance and need to be taken into consideration when formulating theories of calcium influx into the mineralizable matrix of the respective tissues.     

Using 96-well tissue culture polystyrene plates and a fluorescence plate reader as tools to study the survival and inactivation of viruses on surfaces

A method for studying the behavior of viruses on surfaces has been developed and is illustrated by determining the temperatures that inactivate adsorbed viral hemorrhagic septicemia virus (VHSV) and the concentration of 1-propanol that disinfected surfaces with adsorbed VHSV and chum salmon virus (CSV). VHSV is a rhabdovirus; CSV, a reovirus, and they were detected with two fish cell lines, EPC and CHSE-214, respectively. When polystyrene tissue culture surfaces were incubated with virus, rinsed, and left to dry, they still supported the attachment and spreading of cell lines and after 7 days these cells showed the characteristic CPE of the viruses. Thus cells appeared to be infected directly from surfaces on which viruses had been adsorbed. Applying this property to 96-well plates allowed duplicate surfaces to be examined for their infectiousness or support of CPE. For each treatment 80 replicate surfaces in a 96-well plate were tested at one time and the results expressed as the number of wells showing CPE. VHSV adsorbed to polystyrene was inactivated by drying in the dark at temperatures above 14 °C, but remained infectious for at least 15 days of drying at 4 °C. For chemical sterilization of polystyrene surfaces with adsorbed virus, disinfection was achieved with 1-propanol at 40% for VHSV and at 60% for CSV. As CPE can be conveniently monitored in 96-well plates with a fluorescence plate reader, this method can be used to rapidly evaluate a variety of treatments for their ability to inactivate surface-bound viruses.     

Use of Low Temperature Scanning Electron Microscopy to Observe Frozen Hydrated Specimens of Nematodes

Frozen hydrated specimens of Pratylenchus agilis and dauer larvae of Steinernema carpocapsae were observed with low-temperature field emission scanning electron microscopy. This new technique provides information about the surface features of nematodes and also allows specimens to be fractured to reveal their internal structure. Furthermore, both halves of fractured specimens can be retained, examined, and photographed either as two-dimensional micrographs or as three-dimensional images for stereo observation (stereology) or quantitative measurements (stereometry). This technique avoids artifacts normally associated with procedures required to prepare nematodes for examination in the transmission and scanning electron microscopes, such as chemical fixation, dehydration, and sectioning or critical point drying.     

Scanning electron microscopic studies of virus-infected cells. I. Cytopathic effects and maturation of vesicular stomatitis virus in L2 cells.

L2 cells infected with vesicular stomatitis virus under single-cycle conditions have been studied by scanning electron microscopy after preparation by the critical point drying technique. Three dimensional images of intact cells show bullet-shaped vesicular stomatitis virus virions budding singly and in radiating clusters both from the plasma membrane between cellular microvilli and from the sides of microvilli. Virus-induced cytopathic effects observed by scanning electron microscopy include intermeshing of microvilli, loss of filipodia which attach cells to the substrate, and rounding up and detachment of infected cells from the substrate.     

A Comparison of Techniques Useful for Preparing Nematodes for Scanning Electron Microscopy

Second-stage juveniles of Meloidogyne incognita were prepared by several different techniques for scanning electron microscopy (SEM). Sequential fixation in the cold (4-8 C) was superior to rapid fixation at room temperature, glutaraldehyde and glutaraldehyde-formalin were better fixatives than formalin alone, and critical point drying with carbon dioxide or Freon gave similar results that were only slightly better than air drying with Freon. Freeze drying sequentially fixed nematodes from 100% ethanol in liquid propane produced the best preserved specimens with the fewest artifacts. Specimens of various free-living and plant-parasitic nematodes were prepared for SEM by freeze drying. This technique was adequate for most genera but unsatisfactory for a few. Although each genus may require a different procedure for optimum preservation of detail, sequential fixation with glutaraldehyde and freeze drying are comparable and often superior to commonly used techniques for preparing nematodes for SEM.     

Egress of varicella-zoster virus from the melanoma cell: a tropism for the melanocyte.

The pathway of envelopment and egress of the varicella-zoster virus (VZV) and the primary site of viral production within the epidermal layer of the skin are not fully understood. There are several hypotheses to explain how the virus may receive an envelope as it travels to the surface of the monolayer. In this study, we expand earlier reports and provide a more detailed explanation of the growth of VZV in human melanoma cells. Human melanoma cells were selected because they are a malignant derivative of the melanocyte, the melanin-producing cell which originates in the neural crest. We were able to observe the cytopathic effects of syncytial formation and the pattern of egress of virions at the surfaces of infected monolayers by scanning electron microscopy and laser-scanning confocal microscopy. The egressed virions did not appear uniformly over the syncytial surface, rather they were present in elongated patterns which were designated viral highways. In order to document the pathway by which VZV travels from the host cell nucleus to the outer cell membrane, melanoma cells were infected and then processed for examination by transmission electron microscopy (TEM) at increasing intervals postinfection. At the early time points, within minutes to hours postinfection, it was not possible to localize the input virus by TEM. Thus, viral particles first observed at 24 h postinfection were considered progeny virus. On the basis of the TEM observations, the following sequence of events was considered most likely. Nucleocapsids passed through the inner nuclear membrane and acquired an envelope, after which they were seen in the endoplasmic reticulum. Enveloped virions within vacuoles derived from the endoplasmic reticulum passed into the cytoplasm. Thereafter, vacuoles containing nascent enveloped particles acquired viral glycoproteins by fusion with vesicles derived from the Golgi. The vacuoles containing virions fused with the outer plasma membrane and the particles appeared on the surface of the infected cell. Late in infection, enveloped virions were also present within the nuclei of infected cells; the most likely mechanism was retrograde flow from the perinuclear space back into the nucleus. Thus, this study suggests a role for the melanocyte in the pathogenesis of VZV infection, because all steps in viral egress can be accounted for if VZV subsumes the cellular pathways required for melanogenesis.     

Extracellular matrix of plant callus tissue visualized by ESEM and SEM

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.