Germination and Outgrowth of Single Spores of Saccharomyces cerevisiae Viewed by Scanning Electron and Phase-Contrast Microscopy

Single spores of Saccharomyces cerevisiae were examined during germination and outgrowth by scanning electron and phase-contrast microscopy. Also determined were changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOH-soluble carbohydrates. These studies reveal that development of the vegetative cell from a spore follows a definite sequence of events involving dramatic physical and chemical modifications. These changes are: initial rapid loss in cellular absorbance followed later by an abrupt gain in absorbance; reduction in cell weight and a subsequent progressive increase; modification of the spore surface with concomitant diminution in refractility; elongation of the cell and augmentation of surface irregularities; rapid decline in trehalose content of the cell accompanied by extensive formation of KOH-soluble carbohydrates; and bud formation.     

Scanning Electron Microscopy of Bacterial Colonies

A technique is described for observing bacterial colony growth. Bacillus cereus, B. subtilis, and B. cereus var. mycoides were grown on strips of dialysis membrane layered on nutrient agar. Microcolonies of the organisms on strips were fixed in Formalin vapor in situ; the strips then were removed from the agar and secured to scanning microscope specimen stubs without markedly disturbing the cellular arrangement. Scanning electron micrographs clearly depict morphology of individual cells, as well as the spatial orientation of cells within the colony. This technique is reproducible, adaptable, and simple.     

Critical Point Drying of Liverwort Spores for Scanning Electron Microscopy

Preparation for scanning electron microscopy by the Freon-critical point method prevents the collapse of thin-walled liverwort spores and eliminates desiccation artifacts. The success of the method is demonstrated by stereomicrographs of 1) the thin-walled multicellular spore of Conocephalum conicum (Marchantiales) and 2) the thin-walled Jungermannialean spore of Lophocolea heterophylla.     

Surface Features of Bacillus polymyxa Spores as Revealed by Scanning Electron Microscopy

The surface features of Bacillus polymyxa spores were compared by use of thin sections, carbon replicas, and the scanning electron microscope. Some features of the characteristic ridges, previously reported in ultrathin sections and carbon replicas of spores of this species, were more clearly revealed with the scanning electron microscope. A three-dimensional image is provided because of the greater depth of focus possible with this instrument. End-on views of B. polymyxa spores readily illustrate the polygonal porelike structure present.     

Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

Germination of Bacillus spores with a high pressure (HP) of ∼150 MPa is via activation of spores'' germinant receptors (GRs). The HP germination of multiple individual Bacillus subtilis spores in a diamond anvil cell (DAC) was monitored with phase-contrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ∼150 MPa and 37°C but individual spores'' germination kinetics were heterogeneous; (ii) individual spores'' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (T_lag) prior to a period of the rapid release (ΔT_release) of the spores'' dipicolinic acid in a 1:1 chelate with Ca²⁺ (CaDPA); (iii) spore germination at 50 MPa had longer average T_lag values than that at ∼150 MPa, but the ΔT_release values at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv) B. subtilis spores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average ΔT_release values ∼15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average T_lag values; and (v) the germination of wild-type spores given a ≥30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for ≥35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ∼150 MPa for ≤30 s is sufficient to fully activate spores'' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.     

Scanning Electron Microscopy of Plant Roots

A glycol methacrylate infiltration and polymerization techniquewas used to prepare clover roots inoculated with Rhizobium forscanning reflection electron microscopy. Root hairs and epidermalcells were coated with many bacteria; some bacteria seemed tobe embedded in the wall surface. Root hair tips were often smoothbut some older root hair surfaces showed a fibrillar meshworkpattern. Small granules c. 0.18 µm diameter were presenton the root hair and epidermal cell walls. The root cap, someroot hairs, and some epidermal cells were covered by an amorphousfilm thought to be the mucigel.     

Scanning Electron Microscopy of Thermoplasma acidophilum

The scanning electron microscope was utilized to observe the morphology of the thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum. Upon examination of the surface morphology, the size and shape of this unusual mycoplasma revealed its similarity to the other mycoplasmas that have been investigated.     

Transmission Electron Microscopy of Tissue Prepared for Scanning Electron Microscopy by Ethanol-Cryofracturing

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of sections cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

Correlative Photoactivated Localization and Scanning Electron Microscopy

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.     

Scanning Electron Microscopy of Ascospores of Schwanniomyces

Ascospores of the four recognized species of Schwanniomyces were examined by scanning electron microscopy. Spores of S. alluvius, S. castellii, and S. occidentalis, which were essentially identical, had abundant, long protuberances and wide, thin equatorial rings. The two known strains of S. persoonii differed from the other species as well as from each other. One strain had spores with a wide ring but only a few short protuberances; spores from the second strain were covered with craterlike depressions and had a narrow ring. Also examined were spores of Schwanniomyces hominis (=Saccharomyces rosei) which lacked a ring and were covered with short irregularly shaped protuberances, a finding consistent with the morphology of spores from other strains of S. rosei.     

Hexamethyldisilazane for Scanning Electron Microscopy of Gastrotricha

We evaluated treatment with hexamethyldisilazane (HMDS) as an alternative to critical-point drying (CPD) for preparing microscopic Gastrotricha for scanning electron microscopy (SEM). We prepared large marine (2 mm) and small freshwater (100 μm) gastrotrichs using HMDS as the primary dehydration solvent and compared the results to earlier investigations using CPD. The results of HMDS dehydration are similar to or better than CPD for resolution of two important taxonomic features: cuticular ornamentation and patterns of ciliation. The body wall of both sculpted (Lepidodermeila) and smooth (Dolichodasys) gastrotrichs retained excellent morphology as did the delicate sensory and locomotory cilia. The only unfavorable result of HMDS dehydration was an occasional coagulation of gold residue when the solvent had not fully evaporated before sputter-coating. We consider HMDS an effective alternative for preparing of gastrotrichs for SEM because it saves time and expense compared to CPD.     

Cryo-Preservation of Roots for Scanning Electron Microscopy

Fully hydrated roots can be examined in the scanning electronmicroscope after cryo-preservation. Shrinkage associated withdehydration by freeze-drying or critical point drying, to whichroot hairs and secreted mucigel are particularly vulnerable,is avoided. Roots, Lepidium sativum, scanning electron microscopy, cryo-preservation, fully hydrated     

Scanning Electron Microscopy of Intact Trichoderma Colonies

Scanning electron microscopy revealed a clear distinction between hyphal types and enabled early detection of hyphal initiation. Stages in the photoinduced differentiation in Trichoderma leading to conidiation could thus be studied.     

Scanning Electron Microscopy of Freeze-dried Protein Foams

Two proteins of low molecular weight, which bind cadmium and are rich in cysteine were isolated from kidneys of the striped dolphin, Stenella coeruleoalba, and identified as metallo-thioneins I and II. The two isoforms closely resembled horse renal isometallothioneins both in chromatographic behaviors and chemical compositions. The molecular weights of performic acid-oxidized proteins were estimated to be 6,800. Twenty to 21 cysteine residues and about 6 g-atoms of heavy metals (Zn, Cd, Cu, and Hg) were present per mole of each isomer.     

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.     

Scanning Electron Microscopy of Intact Colonies of Microorganisms

Colonies of S. mutans OMZ61, Streptococcus sp. D182, Staphylococcus aureus Oxford NCTC 6571, and Candida albicans type A, MRL 3153 were grown on various media. Cubes of agar bearing two to three colonies were excised and processed for scanning electron microscopy. The characteristic shape of the colonies was seen when examined at low magnifications. At a magnification of 2,000 diameters, the arrangement of individual organisms within the colonies was observed. Plano-convex colonies consisted of uniformly distributed organisms, whereas S. mutans colonies presented a more complex arrangement possibly associated with the production of extracellular polysaccharides. Certain colonies were totally or partially covered by an adherent film through which the outline of the organisms could be distinguished.     

Fixation of Platelets for Scanning and Transmission Electron Microscopy

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

Scanning Electron Microscopy of Plasma Etched Implant Specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 µl;m histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.