Improved Method for Determination of Respiring Individual Microorganisms in Natural Waters

A method is reported that combines the microscopic determinations of specific, individual, respiring microorganisms by the detection of electron transport system activity and the total number of organisms of an estuarine population by epifluorescence microscopy. An active cellular electron transport system specifically reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, which is recognized as opaque intracellular deposits in microorganisms stained with acridine orange. In a comparison of previously described sample preparation techniques, a loss of >70% of the counts of INT-reducing microorganisms was shown to be due to the dissolution of INT-formazan deposits by immersion oil (used in microscopy). In addition, significantly fewer fluorescing microorganisms and INT-formazan deposits, both ≤0.2 μm in size, were found for sample preparations that included a Nuclepore filter. Visual clarity was enhanced, and significantly greater direct counts and counts of INT-reducing microorganisms were recognized by transferring microorganisms from a filter to a gelatin film on a cover glass, followed by coating the sample with additional gelatin to produce a transparent matrix. With this method, the number of INT-reducing microorganisms determined for a Chesapeake Bay water sample was 2-to 10-fold greater than the number of respiring organisms reported previously for marine or freshwater samples. INT-reducing microorganisms constituted 61% of the total direct counts determined for a Chesapeake Bay water sample. This is the highest percentage of metabolically active microorganisms of any aquatic population reported using a method which determines both total counts and specific activity.     

Correlation between Light and Electron Microscopic Observations and Identification of Mycoplasmalike Organisms using Consecutive 350 nm Thick Sections

Serial sections of 350 nm thickness were used to make a correlation between electron and light microscopic observations. While thionin-acridine orange staining gave a positive result to detect abnormal sieve tubes of phyllody affected Phlox drummondii Hook, when observed under light microscope, the same cells revealed the presence of typical mycoplasmalike organisms (MLOs) in electron microscopic examination. Advantage of 350 nm thick sections in electron microscopy, and the utility of the technique in MLO detection have been discussed.     

Improved Microautoradiographic Method to Determine Individual Microorganisms Active in Substrate Uptake in Natural Waters

We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with ³H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (³H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Correlative light/electron microscopy for the investigation of microbial mats from Black Sea Cold Seeps

In several fields of cell biology, correlative microscopy is applied to compare the structure of objects at high resolution under the electron microscope with low resolution light microscopy images of the same sample. It is, however, difficult to prepare samples and marker systems that are applicable for both microscopic techniques for the same specimen at the same time. In our studies, we used microbial mats from Cold Seep communities for a simple and rapid correlative microscopy method. The mats consist of bacterial and archaeal microorganisms, coupling reverse methanogenesis to the reduction of sulfate. The reverse methanogenic pathway also generates carbonates that precipitate inside the mat and may be the main reason for the formation of a microbial reef. The mat shows highly differentiated aggregates of various organisms, tightly interconnected by extracellular polysaccharides. In order to investigate the role of EPS as adhesive mucilage for the biofilm and as a precipitation matrix for carbonate minerals, samples were embedded in a hydrophilic resin (Lowicryl K4 M). Sections were suitable for light as well as electron microscopy in combination with lectins, either labeled with a fluorescent marker or with colloidal gold. This allows lectin mapping at low resolution for light microscopy in direct comparison with a highly resolved electron microscopic image.     

Preparation of Drosophila mitotic chromosomes for electron-microscopic studies]

A method of chromosome spreading on microscopic slides was modified for electron microscopy of metaphase chromosomes in Drosophila tissues. The slides covered with an electron transparent film were plasmochemically modified to make them hydrophilic. A piece of fixed tissue was macerated in 60% propionic acid before spreading chromosomes over the slide. The parts of preparation selected under light microscope for electron microscopic examination were cut and peeled of the slide to the top of a water drop. It was shown that the resolution of chromosomal structures was significantly higher than seen under optical microscope, but lower than in serial sections.     

CD56(NCAM) antigen in glandular epithelium of human thyroid: light microscopic and ultrastructural study

CD56 antigen, an isoform of the neural cell adhesion molecule (NCAM) was previously found by us in human thyroid by APAAP immunohistochemistry in light microscopy on frozen tissue sections. In the current study, it was attempted to trace the antigen in question using another light microscopic immunohistochemical procedure and to validate the results at the ultrastructural level. For light microscopy, cryostat sections of 12 surgical samples of human thyroid were subjected to ABC (preformed avidin-biotin-peroxidase complex) method. For immunoelectron microscopy, immunoperoxidase reaction was carried out on prefixed, small thyroid tissue blocks. Following preliminary inspection of semithin sections, ultrathin sections were examined in the transmission electron microscope. ABC reaction revealed distinct specific CD56 staining of thyrocyte cell membranes. The staining was weak or absent in thyroid papillary carcinoma cells. The results were confirmed in semithin sections by indirect immunoperoxidase. The latter reaction in ultrathin sections at the ultrastructural level has shown that specific reaction product was confined to free and lateral surfaces of thyroid follicular cells. Endothelial cell membranes of thyroid capillary vessels were totally devoid of the reaction product. The reaction was weakly positive in thyroid follicular and papilllary carcinomas but absent from medullary carcinoma.     

Identification of enterochromaffin cells in adjacent Epon-embedded sections at light and electron microscopic levels

Summary Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki, Finland.The electron microscopic observations were carried out in the Electron Microscope Laboratory, University of Helsinki.     

Distribution of virus-like Particles in an Oligotrophic Marine Environment (Alboran Sea,Western Mediterranean)

Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude. In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment. Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM). The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses. In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20. VLP (virus-like particle) counts were not significantly correlated (p > 0.001) with chlorophyll a concentration or the abundance of cyanobacteria. However, there was a positive and significant correlation with bacterial abundance (p < 0.001). The analysis of size and morphology of viral particles by TEM and the correlation obtained between the numbers of VLP and bacteria suggest that the majority of the viral particles in the Alboran Sea are bacteriophages. None of the indirect evidence suggested that eukaryotic algae or cyanobacteria were important host organisms in these waters.     

Nitrogen fixation in lakes

Summary Determinations in the open waters of lakes using N¹⁵ as a tracer show that nitrogen fixation is generally associated with the presence of heterocystous blue-green algae and is light dependent. Although nitrogen-fixing blue-green algae tend to be abundant when the concentration of nitrate or ammonia in the water is low, fixation itself is not necessarily inhibited by the presence of these sources of combined nitrogen. The activity of nitrogen-fixing blue-green algae shows a direct relationship to concentration of dissolved organic nitrogen. As a result of the interaction of such factors, nitrogen fixation per unit area of lake surface per year tends to be greatest at an early stage of eutrophication. In relation to the total nitrogen budget of a lake the contribution of biological nitrogen fixation to the income is probably always small but at certain times and in particular water strata it may contribute a major part of the nitrogen assimilated by the phytoplankton.     

Life after treatment: detecting living microorganisms following exposure to UV light and chlorine dioxide

Rapid analytical methods are needed to quantify living microorganisms to determine if ships’ discharged ballast water is in compliance with national and international standards. Traditionally, regrowth assays and microscope counts of stained organisms—which are time-consuming, require expensive equipment, and require extensive staff training—are used to assess microorganisms. The goal of this study was to evaluate other approaches. Both ambient microorganisms from an oligotrophic marine environment and laboratory cultures of marine algae were evaluated following exposure to two types of ballast water treatment: ultraviolet (UV) light and chlorine dioxide (ClO₂). Microorganisms in two size classes (<10 and ≥10 to <50 μm) were quantified using regrowth assays and vital staining, and samples were evaluated using two rapid approaches: (1) chlorophyll a fluorescence and photochemical yield were measured using a pulse amplitude modulated fluorometer and (2) the concentration of adenosine triphosphate (ATP) was measured with a handheld luminometer. The response of microorganisms to UV and ClO₂ was evident in measurements of photochemical yield, as photochemical yield decreased at high doses. However, initial values of photochemical yield were variable and species-specific. Oddly, in some trials, initial fluorescence increased at intermediate UV doses; this phenomenon could lead to overestimation of total biomass. In samples treated with UV light, ATP was not significantly different among any of the doses used; however, concentrations of ATP were significantly lower at the highest dose of ClO₂ than control samples. These results demonstrate that approaches used for ballast water testing can be treatment-specific, and compliance approaches should be validated to determine their utility with the appropriate treatments.     

Morphology,morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma

Summary The host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.The authors wish to thank Christiana Ulness and Andrea Erickson for expert technical assistance and Arnold Schmidt for the operation of the scanning electron microscope. This work was supported by grants from the U.S.P.H.S.: AI 09586, AI 10743, and AI 06720     

Light microscopic morphometric analysis of peroxisomes by automatic image analysis: advantages of immunostaining over the alkaline DAB method.

The feasibility of light microscopic post-embedding immunocytochemistry for morphometry of peroxisomes using automatic image analysis was investigated and compared with the classical alkaline DAB method. Perfusion-fixed rat liver tissue was either embedded in LR White or incubated in the alkaline diaminobenzidine (DAB) medium for cytochemical visualization of catalase. Sections from the LR White-embedded material were incubated with a monospecific antibody against catalase, followed by protein A-gold and silver intensification. Determination of peroxisomal volume density in sections of different thickness revealed that the values increased with section thickness in DAB-stained sections but were unaffected in immunostained preparations. Moreover, the absolute value for volume density of peroxisomes, as determined by light microscopy in immunostained sections, was quite close to the value obtained by analysis of electron microscopic preparations. Finally, morphometric analysis of bezafibrate-induced peroxisome proliferation revealed that the ratio of proliferation obtained by light microscopy in immunostained sections was very close to the results obtained by electron microscopic morphometry. The main advantage of post-embedding immunostaining for light microscopic morphometry is that it restricts the immunocytochemical reaction product to the surface of the section, thus making it independent of section thickness.     

A Method for the Examination of the Same Cell Using Light, Scanning and Transmission Electron Microscopy

A method is described for preparing the same cell from a cytospin preparation for comparative investigation by light microscopy, scanning electron microscopy and transmission electron microscopy. A permanent numbered grid pattern was etched on a glass microscope slide to facilitate cell location in each microscopic mode. Data from one cell or group of cells was thus obtained from three sources. This method provides a useful adjunct to routine cytological diagnosis.     

浮游病毒的电镜观察

直接用戊二醛固定水样中的浮游病毒,通过超速离心使已固定的浮游病毒沉淀到覆有Formvar膜和碳支持膜的铜网上,经醋酸双氧铀染色后,利用透射电镜对湖水中的浮游病毒进行观察．结果可观察到球形、杆状和蝌蚪状等形态各异的浮游病毒颗粒及球形病毒的囊膜子粒、杆状病毒的核衣壳、具有不同尾部的蝌蚪状病毒等的精细超微结构．从而建立了一种简便、快捷和高效研究浮游病毒的电镜方法．     

Light microscopic identification and photography of Golgi impregnated central nervous system neurons during sectioning for electron microscopy.

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin sections of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which part of the neuron and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

Characterization of Subsurface Bacteria Associated with Two Shallow Aquifers in Oklahoma

The bacterial microflora of two shallow aquifers (saturated subsurface zones) in Oklahoma was characterized by direct observation with light and electron microscopy, by plating, and by examination of colony morphology and distribution. Isolated bacterial strains were also examined. Total cell counts varied only slightly (2.9 × 10⁶ to 9.8 × 10⁶ g [dry wt]⁻¹) from sample to sample, whereas colony counts varied widely (6.3 × 10² to 6.5 × 10⁶ CFU g [dry wt]⁻¹). Colony counts on nutritionally rich media were lower than on low-nutrient media, especially in samples from the saturated zone. The variety of colony types growing on nutritionally rich media decreased with increasing depth and saturation. Colony counts of anaerobic bacteria also decreased with depth but were at least 100-fold lower than aerobic counts on most media. Cell morphologies of bacteria grown aerobically on plates included short rods, cocci, and actinomycete-like forms. Direct light microscopic observation of sediments revealed short, rod-shaped, and coccoid bacterial cells; endospores, actinomycete spores, and eucaryotic forms were not observed by light microscopy. Electron microscopic observation of bacteria released from the samples revealed that 85 to 90% of them were coccoid, gram-positive, Arthrobacter-like organisms, some of which were dividing or contained completed division septa; other types of gram-positive and gram-negative bacteria were present in lower numbers. Isolated bacterial strains were able to grow on both nutritionally rich and low-nutrient media. A higher proportion of gram-negative organisms was isolated than gram-positive organisms. Most of the isolates were capable of storing polyphosphate, poly-β-hydroxybutyrate, or polysaccharide. The results of this study suggest that the microbial population of these two shallow aquifers is dominated by aerobic, nutritionally versatile bacteria that can subsist on low concentrations of organic compounds without forming specialized resting cells. Other types of microorganisms, such as facultatively anaerobic bacteria and microeucaryotes, may also be present, but they represent only a small fraction of the microflora.     

Light Microscopic Identification and Photography of Golgi Impregnated Central Nervous System Neurons During Sectioning for Electron Microscopy

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin door of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which put of the and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

Fiber analysis of an insect peripheral nerve: Light vs. electron microscopy

This paper describes the morphology of one of the two major femoral nerve trunks (nerve V of Dresden and Nijenhuis (1958)) of the cockroach Blaberus discoidalis as revealed by light microscopy, scanning electron microscopy and transmission electron microscopy. Fiber analysis of light and electron micrographs of thick and thin sections taken from adjacent areas of the same nerve yield significantly different data about the number and size distribution of the component axons of nerve V. The light microscopic image indicates one example of nerve V contains 2030 individual axons with a mean diameter of 1.4 μ; the electron image indicates the nerve contains 5794 axons with a mean diameter of 0.56 μ. The disparity between the two types of data is due to the large number of very small axons (0.2 μ or less in diameter) that course through the femoral nerve.