One-third of the worldwide sodium chloride (salt) production (about 200 million tons per year) is manufactured in solar saltworks. A solar saltworks is a series of one or more interconnected, shallow concentrating ponds (40 to 70 cm deep) through which seawater flows, evaporates by wind and sun, and deposits sodium chloride on the floors of crystallizer ponds (30 cm to 50 cm deep). In the downstream flow, salts of low solubility (in respect to sodium chloride) precipitate, with calcium carbonate first dropping out near three times, and calcium sulfate (gypsum) first appearing near four times seawater salinity. Shortly before reaching saturation with sodium chloride (about S.G. 1.214), the brine flows into crystallizer ponds (crystallizers), where evaporation continues until 5 to 20 cm of sodium chloride is deposited on the floors. The supernatant liquid, rich in compounds of calcium, magnesium, chloride, and sulfate, is removed, and the salt is harvested, washed, stockpiled for a time to decrease contaminants, and marketed.

A seldom studied and poorly understood feature of cyanobacterial growth and development is the formation of ordered macroscopic structures from microscopic cells, trichomes, and filaments. The forms range from nostoc balls and layered mats of comparatively simple structure to more complex erect tree-like (dendroid) macro-structures. Using macrophotography, time lapse micro-cinematography, light and scanning electron microscopy of Nostoc species in pure culture, it has been possible to show how motility, adhesion and aggregation of photo-induced motile trichomes (hormogonia) result in the macro-morphogenesis of dendroid forms.

Nostocacean development proceeds by the differentiation of cells in a cyclic sequence of stages including: long chains of cells in heterocystous filaments, motile hormogonia, and large rounded cells termed the aseriate phase. When grown from localized inocula on solid media, sequential development recycles across the agar surface of the culture. Different strains and species vary in the form and duration of each stage and the photo-response of hormogonia.

Automobile air conditioning systems (ACS) might be considered an extreme environment for many microorganisms. Organisms surviving and proliferating in these systems may be presented with temperature changes ranging from subzero to over 140°F, water activity from saturation to dryness and a nutrient complexity including varying levels of hydrocarbons. Microbial communities develop in ACS and sometimes proliferate to the extent of massive colonization and production of objectionable odors.

In a few instances microorganisms emanating from ACS have been associated with hypersensitivity pneumonitis and other allergic reactions. Previously we demonstrated that foam insulation and glues, in particular, on ACS insulations were colonized by fungi such as Aspergillus, Aureobasidium, Cladosporium, and Penicillium. Such fungi often are implicated in colonization of similar substrates in buildings categorized with the sick building syndrome.

A total of 42 ACS from the United States, Europe and Central America have been examined by cabin air sampling culture swab, direct microscopy or a combination of these techniques.

The small (0.5-0.75 um) Clostridial endospores are highly resistant to solvents, acids, bases, radiation, heat, cold, pressure, vacuum and most sporicides. C.difficile causes antibiotic induced pseudomembranous colitis, and is responsible for almost 20% of nosocomial diarrheas in todays hospitals. These endospores have been found on hospital floors, sheets and bedding, as well as on the hands and white coats of hospital personnel.

Our previous studies revealed that these endospores attach to agar by using a two phase attachment process. To examine if this mechanism for spore attachment is also responsible for C.difficile pathogenesis in the human colon, spore attachment and invasion of monolayers of human colon carcinoma cells (Caco-2 cells) using two strains of C.difficile,(ATCC 43594 an isolate from a patient with pseudomembranous colitis [FIG.1A] and ATCC 9689 a clinical microbology laboratory quality control strain) was studied. Cell monolayers grown on 6mm coverslips were inoculated with 10 ul of spore suspension (0.5 McFarland turbidity in sterile water),

Rickettsias are small, gram-negative Alpha Proteobacteria that are, with one exception, obligate intracellular parasites. They are found in a wide range of arthropods. Some of these bacteria are arthropod-vectored disease agents of vertebrates. For example, rickettsias are the causative agents of human typhus and Rocky Mountain spotted fever. Others, such as Wolbachia, are important manipulators of their arthropod host's reproductive capabilities and are not currently known to be disease agents of vertebrates.

Research on Wolbachia and related rickettsias has increased dramatically in recent years. Most of the attention has been focused on insects, although there are occasional reports of rickettsiae being found in isopods and mites. Most recently, the use of molecular techniques has established that Wolbachia is widespread in spider and predatory mites. However, Wolbachia or other rickettsias have not been demonstrated ultrastructurally in the spider mites. This report documents the presence of a rickettsia in the ovary of the adult female twospotted spider mite, Tetranchus urticae Koch, an economically important pest of ornamental and crop plants.

In the yeast Saccharomyces cerevisiae, [PST] acts as an epigenetic modifier of translation termination efficiency. [PSI+] can be passed through generations of yeast cells via changes in protein conformation rather than changes in DNA or RNA, and has thus been referred to as a yeast prion. The [PSI+] determinant is the Sup35 protein. Sup35 can exist in two states - soluble and insoluble. Soluble Sup35 functions in translation termination, but when insoluble, stop codons are read through, resulting in incorrect protein products.

Sup35 is composed of three distinct domains, N, M, and C. The N region is rich in glutamine and asparagine and is required for the [PST] phenotype to exist. M is a highly charged domain, and no specific function has been assigned to it. C is essential in yeast, as it is responsible for translation termination. The insoluble form of Sup35 has characteristics reminiscent of other prion proteins - in vitro it binds to the dye Congo Red and it exhibits apple green birefringence in polarized light.

Economically important insects include pests of plants, animals and stored products as well as insects produced commercially (honey bees, silkworms, insects for fish bait and food for birds and zoo animals). Other insects are produced in large numbers for experimental purposes, biological control of insect pests and weeds and the production of sterile insects for population suppression. Insect viruses may affect morphology, physiology and behavior, often reducing longevity and reproductive potential. Thus insect viruses can be used as biological control agents of pest insects. Insect viruses may also interfere with the production or function of biological control agents (e.g., parasitoids and predators) and insects used for research purposes. Therefore, it is advantageous to screen commercial and research colonies and imported insects for viruses.

Electron microscopy of negatively stained specimens (NS EM) offers a relatively rapid and inexpensive means of screening populations of insects for the presence of viruses or viruslike particles and to monitor the progress of virus control programs.

Previously, we uncovered evidence of an infectious agent, Chlamydia pneumoniae, associated with sporadic Alzheimer's disease (AD). We demonstrated that C. pneumoniae was present in 90% of brain materials examined from AD patients, compared with 5% in age-matched, non-AD patients. We further showed that microglia and astroglia in the CNS are host cells for the bacterium. RT-PCR analyses indicated that the bacterium is metabolically-active in the brain and could be cultured in a human monocyte cell line (THP-1). Importantly, we showed that the organism is concentrated in areas of AD-neuropathology also containing tau and beta-amyloid. The present study was designed to investigate the effects of C. pneumoniae infection on beta amyloid production in human monocytes (THP-1), astroglial (CCF-STTG1), epithelial (Hep 2), and endothelial (HBMEC) cell lines. We investigated the host pathogen relationship with C. pneumoniae and whether entities characteristic of AD neuropathology such as betaamyloid are associated directly with this infectious agent.

The purpose of this work was to examine, by electron microscopy, the mechanism of bacterial binding to oral epithelium in patients with stomatitis using ruthenium red in combination with osmium tetroxide.

Bacteriological semi-quantitative estimation of smear from the palatal mucosa was performed in patients with stomatitis and only patients with viridans Streptococci were further included in the present investigation. Biopsies were taken from the inflamed keratinizing mucosa under regional block anesthesia in agreement with Vancouver guidelines 1978. Control samples were fixed with 1.2% glutaraldehyde (buffered at pH 6.5 with 0.1 M sodium cacodylate) for 2 h at 4° C. Postfixation was performed with 1% osmium tetroxide (buffered at pH 6.5 with 0.1 M sodium cacodylate) for 2 h at 4° C. The other samples were fixed and postfixed by the fixatives mentioned before with addition of 1.6 % ruthenium red in each case.

The eastern coast of north central Florida is home to a habitat unique in the southeastern United States. Some areas of the north to north-central Florida beaches consist of outcroppings of coquina, a beach rock formed by shell fragments and sand held together by a calcareous cement. The major constituents of coquina are shell fragments of the surf clam Donax variabilis, while fragments of Area sp. and Dinocardium sp. are also generally present. These same shell fragments are also found loose comprising most of the beach “sand” in this area. The coquina serves as a substrate for a variety of attatched seaweeds and fouling organisms. Microalgae and cyanophytes of interest to this study can be classified as either epilithic or endolithic. Epilithic microorganisms inhabit the surface, using the rock primarily as a site of attatchment. The endolithic microorganisms are those algae and cyanophytes that actively bore into the coquina rock's shell fragments.

Cryo techniques of specimen preparation have become the standard for cytological studies of biological specimens. From the pioneering work of Feder and Sidman, and Zalokar, the now ubiquitous application of freeze substitution for transmission electron microscope studies of fungi was a direct result of work reported in 1979. Since then, cryopreparative methods have also become the standard for SEM studies and may, for certain purposes, replace conventional methods of fixation for light microscopy as well. Undoubtedly, there are instances where non-cryo methods might be preferred, or where comparisons of results using cryo vs. chemical fixations can provide unique information. On the whole, however, the advantages offered to mycologists and plant pathologists by cryo fixation over any chemical methods are many, and include (a) the opportunity to preserve specimens in a non-aqueous environment, (b) preservation of labile structures such as certain organelles, extracellular matrices, or various cellular content such as ions or that of vacuoles, (c) preservation of cells in a more life-like state,

Allomyces macrogynus is a zoosporic fungus (Chytridiomycota) that exhibits a true alternation of generations between the diploid and haploid thalli. During the diploid stage in the life cycle, sporothalli are produced which are capable of forming asexual zoosporangia containing a multinucleate protoplast bound by a single plasma membrane and cell wall. Though able to remain in a quiescent, coenocytic state for an extended period of time, under certain environmental conditions the cytoplasm of mature zoosporangia is cleaved and gives rise to numerous uninucleate, uniflagellate zoospores within 40 to 50 min. Previous studies of cleavage membrane development m Allomyces zoosporangia have been conducted using light microscopy and transmission electron microscopy (TEM). Despite these investigations, our understanding of cytokinesis in zoosporangia of Allomyces and other zoosporic organisms remains incomplete. This is due, in part, to the difficulty of detecting cleavage elements using standard light microscope optics (i.e., phase contrast, differential interference contrast) and because ultrastructural analysis often provides limited temporal and spatial detail and may suffer from artifacts of sample preparation.

Low temperature, low voltage field emission scanning electron microscopy (cryoSEM) allows brilliant imaging of superbly preserved biological material without suffering the specimen destruction inherent in conventional SEM techniques. Confocal laser scanning microscopy (CLSM) allows observation of fluorescence-labeling within host tissues, utilizing the optical sectioning capability of the CLSM to construct composite stereo images of specimens in three dimensions. These technologies were invaluable in elucidating the septoria tritici blotch infection process on wheat, caused by Mycosphaerella graminicola (anamorph Septoria tritici).

Seedlings inoculated with M. graminicola were incubated for the first 48 h in a dew chamber with constant misting, 20°C days and 16°C nights, then transferred to a growth chamber under the same temperature conditions at 90% RH until development of mature pycnidia, visible macroscopically at 11 days post-inoculation. Samples for cryoSEM were attached to specimen blocks with Histo Prep (Fischer Scientific, Orangeburg, NY) and frozen by plunging blocks into liquid nitrogen slush.

Specific gene products of both pathogens and hosts have been implicated as decisive elements during plant pathogenesis. While expression of some of these genes is constitutive, that of others is likely ephemeral and activated only during a particular stage of the interaction. Because the relative timing of expression may be critical, transcription and translation have often been addressed by extracting mRNA and proteins from infected plant tissue. This approach, however, cannot readily detect proteins of low abundance in bulk samples nor offer much useful information on cell-cell interaction. Only a cytological analysis that employs microscopy can resolve the temporal and spatial details of gene expression. Typically, such protein localization studies have required specific antibodies, but these large probe molecules do not diffuse into living or conventionally fixed cells of either fungal pathogens or plant hosts. For TEM analysis, these permeability-imposed limitations have been reduced by thin sectioning to render accessible antibody binding sites.

The high-pressure freezing instrument exposes a sample to a pressure of 2100 bar, which lowers the freezing point and, as a result, reduces the rate of ice nucleation and ice-crystal growth. The reduced critical cooling rate allows adequate freezing of samples up to 0,3 mm in thickness without using cryoprotectants. Before pressure application, the sample is sandwiched between specimen holders. To optimize heat conductivity and to avoid damage by the high pressure impact, the free space inside the specimen holders and within the sample has to be filled with liquid. This means that plant leaves need to be infiltrated to remove gas from the intercellular space. We have used water, 3-8% methanol in water, 1-hexadecene or heptane as infiltration medium. Subsequently, samples were freeze substituted in unhydrous acetone with 2% Os04 for 24 h at -90°C. Samples were slowly warmed up to 4°C and embedded in Unicryl, or warmed up to room temperature and embedded in epoxide resin.

Since its initial use to fix fungal hyphae (1), plunge freezing followed by freeze substitution has become the “gold standard” for TEM studies of fungal hyphae and spores. In this presentation we discuss results we have obtained using plunge freezing and freeze substitution to fix various types of spores produced by plant pathogenic fungi. Examples include basidiospores and aeciospores of rust fungi, teliospores of rust and smut fungi and conidia of a variety of ascomycetes and deuteromycetes.

Generally speaking, plunge freezing followed by freeze substitution usually yields best results when spore diameter does not exceed 10-15 μm. However, in a number of cases we (2) have obtained good to excellent results with much larger spores. A key to good freezing appears to be direct exposure of spores to liquid propane during plunging. We have had best results by placing spores on small pieces of dialysis membrane and then plunging these membranes. In most cases the spores remain attached to membranes throughout the freezing, substitution, infiltration and embedment procedures.

Determining the length of the various stages of the nuclear cycle and correlating nuclear cycle events with particular aspects of cell morphology generally require the use of autoradiography. The most useful label is tritiated thymidine, because it is incorporated only into DNA. Unfortunately fungi, unlike most other organisms, do not take incorporate thymidine from medium. In the past, microspectrophotometry has been used to study fungal nuclei, but this technique measures nuclei one at a time. Recent advances in image capture and analysis software have made it possible to use the technique of photomicrodensitometry to quantify DNA in nuclei stained with fluorochromes that bind quantitatively to DNA. We are using this method to study nuclear cycle events in cells of the pathogenic fungus Ustilago maydis.

The basidiomycete fungus Ustilago maydis causes corn smut disease. This fungus is dimorphic—in addition to growing by filaments in corn plants, it can exist saprobically in a yeast-like budding stage.

Entomosporium mespili has emerged as a significant pathogen of red tip (Photinia × fraseri), a popular and widely grown ornamental in the southeastern United States. This fungal pathogen produces its distinctive multi-celled, insect-like asexual spores or conidia (Fig. 1) in structures known as acervuli (Fig. 2) that rupture the surfaces of infected leaves. This study examines the fine structure of these conidia using a combination of scanning and transmission electron microscopy. In the case of transmission electron microscopy, conidia were prepared for study using either plunge freezing or high pressure freezing followed by freeze substitution.

Each mature conidium of E. mespili consists of four to six cells (Fig. 1). These include one apical cell and one basal cell and two to four small lateral cells attached to the basal cell. The apical and lateral cells each possess a long, slender appendage. Excluding these appendages, the length of a mature conidium is usually 20-24μm while the diameters of the apical and basal cells are 8-11μm and 6-9μm respectively.

Cryofixation is becoming more widely used to study host-pathogen relationships in fungal diseases of plants. This presentation describes results we have obtained using high pressure freezing and freeze substitution to study powdery mildew disease of poinsettia ﹛Euphorbia pulcherrima) caused by Oidium sp.

Approximately 0.5 mm leaf disks bearing sporulating colonies of Oidium sp. were excised and placed in a 15% dextran solution contained in brass planchets. Samples were frozen using a Balzer's HPM 010 High Pressure Freezing Machine and substituted according to the procedures of Hoch.6 Thin sections of embedded leaves were cut using a diamond knife, collected on gold slot grids, and placed on formvar-coated racks. Sections were poststained with uranyl acetate and lead citrate and examined using a Zeiss EM 902A transmission electron microscope.

Outstanding preservation of haustoria, the specialized nutrient-absorbing structures produced in host epidermal cells by Oidium, was obtained. Both young, unlobed (Fig. 1) as well as mature, highly lobed (Fig. 2) haustoria were observed.

There are number of dwarf mutants in maize such as d8, dl and nal. The nana plant 1 (nal/nal) is characterized as short erect dwarf; the phenotype does not respond to treatment of giberellins. The recessive nal allele is located on the long arm of chromosome 3 (3L-113). In this study, we report the abnormal development of the stem vasculature and the lack of clearly defined nodes.

Maize (Zea mays L., var. Ohio 43 and nal mutant) plants were grown in 1999 summer nursery at the fieldstation of University of Western Ontario, London Ontario, Canada. Plants at approximately 2 months old were fixed in 1:3 acetic acid/ EtOH. Multiple changes of fixative were made for a period of a month to remove as much pigmentation as possible. The specimen was then washed thoroughly, stained in diluted Schiffs reagent (10%), washed in 0.1M metabisulphate solution (with 1 ml of 1 N HC1 per liter), dehydrated in EtOH and cleared in methyl salicylate.