Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.     

Optimization of an Acridine Orange–Bisbenzimide Procedure for the Detection of Apoptosis-Associated Fluorescence Colour Changes in Etoposide-Treated Cell Cultures

This study was initiated in order to investigate the possibility of improving fluorescence microscopy as a method for evaluating apoptosis in cells by combining two fluorescent dyes with different staining characteristics. Cells were vitally stained with bisbenzimide (1.3 microM) and Acridine Orange (6.6 microM) and observed using the following filter configuration: excitation 380 nm, beamsplitter 395 nm and longpass filter 397 nm. Control cells exhibited clear blue fluorescent nuclei and red fluorescing lysosomes. In cells treated with etoposide to induce apoptosis, two distinct occurrences were observed: a change in the spectrum of emitted light from bisbenzimide bound to the nuclear region and an increase in lysosomal Acridine Orange fluorescence. The two occurrences together permit a more unbiased detection of apoptosis than most assays. Only one filter set is required for evaluation and the resulting images can be easily evaluated visually or processed further by image analysis.     

A Toluidine Blue-Membrane Filter Method for the Quantitative Staining of Bacteria

A method for measuring the uptake of toluidine blue by bacteria on membrane filters was developed. Bacteria were filtered out of solution onto a cellulose acetate filter and stained on the filter at 50 C with toluidine blue in citrate-phosphate buffer, pH 4.0. The filter was destained in ethanol, placed on a glass slide and subsequently made transparent in a 1,4-dioxan and cyclohexanone mixture. The absorbance of the stained bacteria on the slide was measured in a spectrophotometer at 590 nm. The uptake of dye by cells of Streptococcus cremoris and Escherichia coli could be explained using the Freundlich adsorption isotherm. Cell concentrations of both these organisms can be determined with this technique.     

A toluidine blue-membrane filter method for the quantitative staining of bacteria

A method for measuring the uptake of toluidine blue by bacteria on membrane filters was developed. Bacteria were filtered out of solution onto a cellulose acetate filter and stained on the filter at 50 C with toluidine blue in citrate-phosphate buffer, pH 4.0. The filter was destained in ethanol, placed on a glass slide and subsequently made transparent in a 1,4-dioxan and cyclohexanone mixture. The absorbance of the stained bacteria on the slide was measured in a spectrophotometer at 590 nm. The uptake of dye by cells of Streptococcus cremoris and Escherichia coli could be explained using the Freundlich adsorption isotherm. Cell concentrations of both these organisms can be determined with this technique.     

Observations of mitochondria and mitochondrial nuclei by double staining with rhodamine-123 and DAPI in synchronous cultures ofCatharanthus roseus

Mitochondria in cells ofCatharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle ofC. roseus.     

Filter combinations and light sources for fluorescence microscopy of quinacrine mustard or quinacrine stained chromosomes

Summary The efficiency of various combinations of primary and secondary filters, and light sources for the fluorescence microscopy of chromosomes stained with quinacrine mustard or quinacrine has been studied quantitatively. Using epi-illumination, strong fluorescence could be obtained with a mercury or xenon lamp in combination with two KP 490 short-wave pass interference filters (tilted to an angle of 60° with the excitation beam) as primary filter, and a K 490 as a secondary filter. The combination of a mercury lamp and a narrow band interference filter with a maximal transmission at about 436 nm as a primary filter together with a K 490 secondary filter results in a good visual image contrast, sufficiently strong fluorescence, and a relatively slow rate of fading.     

A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents

A method is described for quantitation of protein in the presence of reducing agents, detergents, and other substances which often interfere with assays of protein in solution. The proteins are applied to Whatman No. 1 filter paper, air-dried, washed with methanol, and then stained with Coomassie brilliant blue G. Following destaining, the paper is air-dried and the protein-bound dye is extracted. Sample absorbance measurements are made in a 96-well plate using an automated microplate reader (600-405 nm) or in a cuvette at 610 nm. This filter paper assay is useful for determining 100 ng to 20 micrograms of protein in the presence of ammonium sulfate, urea, thiol-reducing agents, amino acids, DNA, ionic and nonionic detergents, and acid or base.     

Mallory's Phloxine B-Methylene Blue-Azure II Stain Emphasizes Elastin and Collagen Bundles in Epoxy Embedded Lung

A version of Mallory's phloxine-methylene blue-azure II technique suitable for large epoxy sections is described. Phloxine B (CI. 45410) and a yellow-green interference filter (546-548 nm transmission) combine to give high contrast monochrome images. By comparing light micrographs of lung parenchyma entirely unstained or stained only with phloxine B against electron micrographs of the same material, it is seen that phloxine B emphasizes essentially only elastin and collagen fiber bundles. The technique has produced images useful for investigating lung parenchyma architecture and micromechanics.     

A colorimetric method for the determination of submicrogram quantities of protein

A simple procedure for the measurement of submicrogram quantities of protein is described which can be used without interference from most common reagents. Protein-containing solutions are spotted on glass fiber filters, washed with trichloroacetic acid, and stained with Coomassie blue. The filters are destained, and the protein-bound colorant is eluted and measured in a spectrophotometer at 590 nm. The response is linear to 5 μg of protein per filter, and as little as 0.1 μg per filter can be accurately determined.     

Mallory's phloxine B-methylene blue-azure II stain emphasizes elastin and collagen bundles in epoxy embedded lung

A version of Mallory's phloxine-methylene blue-azure II technique suitable for large epoxy sections is described. Phloxine B (C.I. 45410) and a yellow-green interference filter (546-548 nm transmission) combine to give high contrast monochrome images. By comparing light micrographs of lung parenchyma entirely unstained or stained only with phloxine B against electron micrographs of the same material, it is seen that phloxine B emphasizes essentially only elastin and collagen fiber bundles. The technique has produced images useful for investigating lung parenchyma architecture and micromechanics.     

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.     

Staining of Merkel cells of pig snout epidermis using the uranaffin reaction

The uranaffin reaction (UR) specifically stains neurosecretory (NS) granules of the neuroendocrine system when performed at pH 3.9 and at a 4% concentration of uranyl acetate. Merkel-cell NS granules stained using the UR were found to have a different appearance than granules observed after routine processing. We therefore compared the average values obtained with both methods, examining the maximum diameter, area, form factor, and numerical density of such NS granules. The maximum diameter and area of NS granules were significantly greater (P less than 0.001) in samples stained using a conventional technique (CT) (93.30 nm, 6,411 nm2) that in those stained with the UR (63.95 nm, 2,148 nm2). The form factor of conventionally stained NS granules (0.9) was significantly greater (P less than 0.001) than that of granules stained with the UR (0.7). No significant difference in numerical density was found for the two techniques (CT: 8.11 +/- 2.51; UR: 6.14 +/- 2.38). It was found that the UR is a useful cytochemical marker for NS granules of Merkel cells, and that the ultrastructural morphology and intracellular arrangement of NS granules stained with the UR are different from those revealed using CT.     

Autofluorescent characteristics of Candidatus Brocadia fulgida and the consequences for FISH and microscopic detection

An enrichment culture of Candidatus Brocadia fulgida was identified by three independent methods: analysis of autofluorescence using different microscope filter blocks and a fluorescence spectrometer, fluorescence in situ hybridization (FISH) with anammox-specific probes and partial sequencing of the 16S rDNA, hydrazine synthase hzsA and hydrazine oxidoreductase hzo. The filter block BV-2A (400–440, 470 LP, Nikon) was suitable for preliminary detection of Ca. B. fulgida. An excitation-emission matrix revealed three pairs of excitation-emission maxima: 288–330 nm, 288–478 nm and 417–478 nm. Several autofluorescent cell clusters could not be stained with DAPI or by FISH, suggesting empty but intact cells (ghost cells) or inhibited permeability. Successful staining of autofluorescent cells with the FISH probes Ban162 and Bfu613, even at higher formamide concentrations, suggested insufficient specificity of Ban162. Under certain conditions, Ca. B. fulgida lost its autofluorescence, which reduced the reliability of autofluorescence for identification and detection. Non-fluorescent Ca. Brocadia cells could not be stained with Ban162, but with Bfu613 at higher formamide concentrations, suggesting a dependency between both parameters. The phylogenetic analysis showed only good taxonomical clustering of the 16S rDNA and hzsA. In conclusion, careful consideration of autofluorescent characteristics is recommended when analysing and presenting FISH observations of Ca. B. fulgida to avoid misinterpretations and misidentifications.     

Conjugates of Chitinase with Fluorescein Isothiocyanate or Lissamine Rhodamine as Specific Stains for Chitin In Sztu

The fluorescent chitinase technique is based on the specific affinity of the enzyme for its substrate and applicable when an enzyme can be coupled with a fluorescent dye. Fluorescent chitinase specifically stained chitinous structures in several fungi and an insect, but failed to stain other polysaccharides in bacterial and algal cell walls. Freezing-microtome sections of Drosophila and fungal mycelia 6 μ thick were fixed in acetone for 5 min, then stained and mounted in fluorescent chitinase. Staining of smears of unsectioned fungal material required 5 min in absolute acetone, 5 min in 95% ethanol-1 N aqueous acetic acid (1:1), 10 min in 0.2 M phosphate buffer, PH 5.7, 1 sec in enzyme-dye conjugate, and 10 min in carbonate-bicarbonate buffer (0.2 M, pH 10.7, for chitinase-FITC; pH 7.6, for chitinase-LRBC). Preparations are viewed microscopically with ultraviolet light.     

Alcian Blue and Colloidal Iron Staining Methods Adapted to Filter Paper Electrophoresis

Filter paper moistened with solutions used in electro-chromatography was spotted with 0.5-1.0μl of solutions of mucopolysaccharides and allowed to air dry. Substances tested with respect to their staining reaction were as follows: (a) From commercial sources: hyaluronic acid, heparin, chondroitin sulfate, ovomucoid and gastric mucin, (b) From natural sources: blood serum, saliva, tears, vitreous filtrate and aqueous humor. Alcian blue was found to be a good general stain for mucopolysaccharides and for locating such material on filter paper, especially when more specific means were used subsequently for identifying the kind of mucopolysaccharide present. Staining by colloidal iron of materials on filter paper was similar to that by the periodic acid-Schiff reaction. However, heparin and chondroitin sulfate were not stained by iron when on filter paper but were stained when placed on glass slides.     

Effects of Sunlight on Stained Sections Mounted in Various Media

Sections stained with haematoxylin and eosin showed maximum optical density (OD) at 536 nm, with a second peak at 600 nm. Sections stained with only eosin showed a peak at 536 nm, whilst those stained with haetnatoxylin showed a peak at 600 nm. Reduction in OD at these wavelengths was used to estimate fading of the staining. Direct sunlight reduced the OD of sections mounted in 22 different mounting media by 14 to 64% at 536 nm and 12 to 51% at 600 nm.     

A dye binding method for measurement of total protein in microalgae

Protein is a large component of the standing biomass of algae. The total protein content of algae is difficult to measure because of the problems encountered in extracting all of the protein from the cells. Here we modified an existing protein assay to measure total protein in microalgae cells that involves little or no extraction of protein from the cells. Aliquots of fresh or pretreated cells were spotted onto filter paper strips. After drying, the strips were stained in a 0.1% (w/v) solution of the protein stain Coomassie Brilliant Blue R-250 for 16 to 24 h and then destained. The stained protein spots were cut out from the paper, and dye was eluted in 1% (w/v) sodium dodecyl sulfate (SDS). Absorbance at 600 nm was directly proportional to protein concentration. Cells that were recalcitrant to taking up the dye could be either heated at 80°C for 10 min in 1% SDS or briefly sonicated for 3 min to facilitate penetration of the dye into the cells. Total protein measured in Chlorella vulgaris using this method compared closely with that measured using the total N method. Total protein concentrations were measured successfully in 12 algal species using this dye binding method.     

Ultrastructure of Klebsiella O3 lipopolysaccharide isolated from culture supernatant: structure of various uniform salt forms

Various uniform salt forms of Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant were prepared as follows. Basic materials present in KO3 LPS were rigorously removed by electrodialysis and the electrodialyzed KO3 LPS was neutralized with NaOH, KOH, NH4OH, Ca(OH)2, tris(hydroxymethyl)aminomethane, or triethylamine. The ultrastructure of the uniform salt forms of KO3 LPS was examined using preparations stained with uranyl acetate. The sodium, potassium, ammonium, and trisaminomethane salt forms were structurally very similar to the natural form of KO3 LPS which consisted of a mixture of flat ribbon-like structures (average width of 16 nm and average thickness of 7 nm) and spheres with various diameters, both covered with fine hairy structures. When KO3 LPS was converted to the triethylamine salt form, the ribbon-like structures were disrupted into very small granules (7-9 nm X 9-15 nm). The calcium salt form consisted of particles and rods of various sizes and ribbon-like structures which were markedly extended (maximum width of 50 nm) and presented irregular shapes. When converted to the calcium salt form, the ribbon-like structures were extended and eventually divided into particles and rods. For reasons still unknown, the sodium salt of KO3 LPS was mostly stained positively with uranyl acetate in contrast to the natural form and the other uniform salt forms which were always negatively stained. In the positively stained preparation of the sodium salt form, it was clearly shown that the ribbon-like structures consisted of a bilayer.     

Analysis of the crystal arrangement in collagen fibrils of mineralizing turkey tibia tendon

Summary Mineralized pieces of tendons from the tibio-tarsus of turkeys were (i) shock-frozen, freeze-dried, embedded and cut without staining, or (ii) fixed, embedded and stained after sectioning. Micrographs were taken with an electron microscope on longitudinally cut sections. The center-to-center distances of neighboring apatitic needles within collagen fibrils were measured. For shock-frozen and freeze-dried specimens, the average of these distances is 4.7 nm and the most frequent value 4.2 nm; for fixed and stained specimens, 3.8 nm and 3.6 nm, respectively. Laser diffraction of the electron micrographs showed a dumbbell-like intensity pattern (two diffuse maxima of intensity on the equator, one on each side of the central spot), giving an average distance of about 6 nm. This value represents the upper range of the direct measurements. The measurements demonstrate that the arrangement of the collagen microfibrils is mainly preserved during mineralization. However, using laser diffraction, distances of 9–11 nm were also observed. Such large distances can also be demonstrated by X-ray diffraction on collagen fibrils stained under special conditions. This may indicate that special conditions of apatitic mineralization or staining may alter the arrangement of the microfibrils.The authors thank the Deutsche Forschungsgemeinschaft for financial support     

Action of UV radiation on the surface of mammalian immunocompetent cells. III. Ultrastructural changes in the glycocalyx of mouse lymphocytes

The outer perimembrane layer of murine spleen lymphocytes were studied with electron microscopy. Mice were treated with short-wave (254 nm) and long-wave (365 nm) ultraviolet radiation in isoeffective lethal doses. The other perimembrane layers were stained with Alcian blue and Ruthenium red. The 254 nm UV treatment decreased the level of dye--sorption by these perimembrane layers, whereas no such effect was obvious after the 365 nm UV treatment.