A new fluorescence technique for staining of mononuclear phagocytes.

A recently developed fluorochrome, 4',6-diamidino-2-phenylindole (DAPI), is used to stain mononuclear phagocytes of the mouse. After addition of heparin, these cells showed a bright yellow outer ring.     

A new and permanent staining method for starch granules using fluorescence microscopy

A fluorescence technique has been developed for observing starch granules in plant tissues. Sections are stained with a mixture of dyes which we have named F.A.S.G.A. from the initials of the Spanish names of its components (fucsina, alcian blue, safranina, glicerina, agua), and viewed by epifluorescence microscopy. The starch granules fluoresce greenish yellow, allowing the degradative state to be observed. Cell structures which do not fluoresce are also differentiated. The stain permits identification of other structures when examined by visible light microscopy and is relatively resistant to fading over time.     

A new staining technique for microfilariae.

A rapid whole mount staining method is described to identify and differentiate microfilariae without elaborate processing. A single solution combining Hoyer's mounting medium and hematoxylin stain facilitates light microscopic examination of nuclei and sheaths of microfilariae. The new technique stains microfilariae adequately in three to seven minutes at 60--64 C making the method preferable to conventional methods that may take as long as 45 to 60 minutes. Lantern heat may be used to heat slides in rural areas with good results.     

A simple fluorescence of staining technique for in situ soil microorganisms.

A simple, rapid straining technique using the magnesium salt of 1-anilino-8-naphthalene sulfonic acid is described. Treatment of soil with an aqueous, membrane-filtered solution (3.5 mg/ml) of the salt causes the soil microorganisms to fluoresce when examined with light from a mercury arc light source.     

A technique for fluorescence microscopy in semithin sections

We describe here a procedure to improve contrast and resolution in fluorescence microscopy of sectioned tissues. Tissue fragments were fixed in ethanol-glacial acetic acid, embedded in diethylene glycol distearate, and semithin sectioned. This method maintains tissue antigenicity while preserving the structure of cells and tissues. The thinness of the sections eliminates scattered and emitted light from tissue structures outside the plane of focus. The procedure is simple and quick, and works excellently with fluorescein-conjugated lectins and antibodies.     

A new type of two-color fluorescence staining for cytology specimens.

A new two-color fluorescence staining technique for cervical cytology specimens is described. To permit application of this staining in automated cytology, techniques for specimen collection and cell preparation giving a sufficient number of well-separated cells on slides were used. The staining consists of a combination of a modified Feulgen-acriflavine procedure for DNA and a primulin or stilbene isothiocyanate staining for protein. This results in a bright yellow nuclear fluorescence and a blue cytoplasmic fluorescence. The staining procedure can be completed in about 90 min and is therefore suitable for routine application. Sequential inspection of the yellow nuclear and blue cytoplasmic fluorescence can be done with the two-wavelength excitation method used in fluorescence microscopy. For the application of this method, special vertical illuminators are now available. These illuminators are provided with quickly interchangeable filter sets permitting consecutive visualization of, for example, only the nuclei in the first image and the whole cell in the second image. This procedure opens new possibilities for rapid image-analysis systems.     

A simple and rapid technique for fluorescence staining of fungal nuclei

A technique for staining fungal nuclei using fluorescence stain Hoechst Dye 33258 in McIlvaine standard buffer of pH 7.26–7.44 is reported. It is a broad-spectrum fungal nuclear staining tool found to be effective onAgaricus bisporus, Alternaria helianthi, Fusarium oxysporum f. sp.lini, Penicillium binellum, Pythium ultimum, Rhizoctonia solani, andSaccharomyces cerevisiae. Conidial nuclei ofAlternaria helianthi, Fusarium oxysporum f. sp.lini, andPenicillium binellum also stained well.     

A new chromosome fluorescence banding technique combining DAPI staining with image analysis in plants

In this study, a new chromosome fluorescence banding technique was developed in plants. The technique combined 4,6-diamidino-2-phenylindole (DAPI) staining with software analysis including three-dimensional imaging after deconvolution. Clear multiple and adjacent DAPI bands like G-bands were obtained by this technique in the tested species including Hordeum vulgare L., Oryza officinalis, Wall & Watt, Triticum aestivum L., Lilium brownii, Brown, and Vicia faba L. During mitotic metaphase, the numbers of bands for the haploid genomes of these species were about 185, 141, 309, 456 and 194, respectively. Reproducibility analysis demonstrated that banding patterns within a species were stable at the same mitotic stage and they could be used for identifying specific chromosomes and chromosome regions. The band number fluctuated: the earlier the mitotic stage, the greater the number of bands. The technique enables genes to be mapped onto specific band regions of the chromosomes by only one fluorescence in situ hybridisation (FISH) step with no chemical banding treatments. In this study, the 45S and 5S rDNAs of some tested species were located on specific band regions of specific chromosomes and they were all positioned at the interbands with the new technique. Because no chemical banding treatment was used, the banding patterns displayed by the technique should reflect the natural conformational features of chromatin. Thus it could be expected that this technique should be suitable for all eukaryotes and would have widespread utility in chromosomal structure analysis and physical mapping of genes.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

Study of constitutive heterochromatin with a new and simplified fluorescence staining technique

A new fluorochrome that preferentially binds itself to the centromeric or the constitutive heterochromatin is described. This stain allows an easy assay, through fluorescence, of the repetitive DNA or bands, supposedly composed of constitutive heterochromatin, in insectivores, rodents and man, without following the in situ hybridization of Pardue & Gall (1970) or the DNA denaturation-renaturation processes of Arrighi & Hsu (1971). The staining patterns with this derivative of a Benzimidazole compound (Hoechst 33258) are induced in the chromosomes without incubation or pretreatment with SSC and are identical to those produced by other techniques. This stain may eventually contribute to elucidating the hitherto unknown molecular mechanisms involved in the relationship between the repetitious DNA sequences and the banding patterns, and to interpreting the mechanisms responsible for the chromosomal rearrangements and aberrations involving the peri-and non-pericentric regions of the chromosomes.Paper read by P.K.S. at the NATO Advanced Study Institute on Comparative Biology of Primates Turin 7–19 June 1972. Supported by the Alexander von Humboldt-Stiftung, Bonn-Bad Godesberg.     

A new technique for preparing biominerals for atomic-force microscopy

Summary We describe a methodology that enables the orienting and cleaving of biominerals to consistently yield low z-axis (height) surfaces suitable for high-resolution imaging by atomic-force microscopy. The methodology permits biominerals to be prepared without chemical fixation, resin embedding, or sectioning, and demonstrates a utility for a variety of silicified and calcified biominerals.     

A new technique for staining catecholic residues in biological samples

This technique for localizing catecholic residues in biological samples is based on the condensation of Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with quinone residues obtained by the oxidation of catechols in the presence of ammonia. The product is a dark pink MBTH-quinone compound. This method is very sensitive and positive to catechol even at the 0.05 microgram level and the final product is chemically stable.     

Contact x-ray microscopy. A new technique for imaging cellular fine structure.

Contact x-ray microscopy potentially allows living, wet cells to be visualized at a resolution of up to 100 A. Furthermore, differential absorption by specific elements permits the study of the distribution of those elements in biological specimens. In contact x-ray microscopy, soft x-rays (10 A to 100 A) pass through a biological sample and expose an underlying x-ray sensitive polymer (resist), producing an image that reflects the photon absorbance within the specimen. The high penetrating power of soft x-ray enables images to be obtained from specimens up to several microns thick. In this paper, the technique is described, some of the areas currently under study are considered, and biological examples of the use of contact x-ray microscopy are given.