Cytophotometric analysis of the chromatin structural conformity in interphase nuclei detected in UV light and by gallocyanine staining

Geometric and optical parameters of chromatin of hepatocyte nuclei have been examined before (UV, lambda = 265 nm) and after gallocyanine staining. Quantitative parameters of the chromatin structure in the same nuclei measured in situ by a scanning microscope-photometer (step size 0.125 micron) before and after staining were equal. Tinctorial properties of chromatin granules (condensed part of the nuclear material) and its diffuse part were different. It is suggested that the difference between granules and the nongranular part of chromatin is not only of optical but also of chemical nature.     

Densitometric determination of nucleic acids in gels after electrophoresis

A technique for the quantitative determining of nucleic acids after electrophoresis by double-wave densitometry in UV spectrum by scanning agar gel along both axes was developed. Optical characteristics of agar and acrylamide-agar gels are given. The range of quantities measured is 6 divided by 30 micrograms of DNA per gel; the standard deviation adjusted to 1 microgram is less than +/- 3%.     

Ultrasensitive staining of nucleic acids with silver

A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm². The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.     

Red-blue staining of hydrolysed nucleic acids in paraffin sections

A previous treatment with 10% HC1 in tetrahydrofuran for 2-3 min at 37° C hydrolyses DNA while substantially preserving RNA in formol-fixed paraffin sections. If this treatment is followed by dyeing with basic fuchsin-thiazine or oxazine mixtures, the basic fuchsin stains DNA, the blue dye cytoplasmic RNA, though nucleolar RNA is not well preserved. A specimen sequence is to treat the hydrolysed section with a mixture of 1% aqueous trimethylthionin (Chroma), 15 ml; 0.1% basic fuchsin (G. T. Gurr), 4 ml; and glacial acetic acid, 1 ml. Stain for 15-30 min, dehydrate in acetone, then pass sections through xylene to polystyrene. The specificity of this stain for cytoplasmic RNA is sharper than that of methyl green-pyronin; hence the technic given can be a useful addition to the standard Unna-Pappenheim procedure.     

Methyl green-pyronin; basis of selective staining of nucleic acids

1. Methyl green stains selectively highly polymerized desoxyribonucleic acid, and fails to stain, to any significant extent, depolymerized desoxyribonucleic acid and ribonucleic acid. 2. Pyronin stains preferentially low polymers of nucleic acid. 3. Triphenylmethane dyes with two amino groups appear to share the selectivity of methyl green. Those with three amino groups are not selective. 4. A stereochemical hypothesis is offered to account for these observations.     

Sensitive determination of microbial growth by nucleic acid staining in aqueous suspension

The determination of cell numbers or biomass in laboratory cultures or environmental samples is usually based on turbidity measurements, viable counts, biochemical determinations (e.g., protein and lipid measurements), microscopic counting, or recently, flow cytometric analysis. In the present study, we developed a novel procedure for the sensitive quantification of microbial cells in cultures and most-probable-number series. The assay combines fluorescent nucleic acid staining and subsequent fluorescence measurement in suspension. Six different fluorescent dyes (acridine orange, DAPI [4',6'-diamidino-2-phenylindole], ethidium bromide, PicoGreen, and SYBR green I and II) were evaluated. SYBR green I was found to be the most sensitive dye and allowed the quantification of 50,000 to up to 1.5 x 10(8) Escherichia coli cells per ml sample. The rapid staining procedure was robust against interference from rRNA, sample fixation by the addition of glutaric dialdehyde, and reducing agents such as sodium dithionite, sodium sulfide, and ferrous sulfide. It worked well with phylogenetically distant bacterial and archaeal strains. Excellent agreement with optical density measurements of cell increases was achieved during growth experiments performed with aerobic and sulfate-reducing bacteria. The assay offers a time-saving, more sensitive alternative to epifluorescence microscopy analysis of most-probable-number dilution series. This method simplifies the quantification of microbial cells in pure cultures as well as enrichments and is particularly suited for low cell densities.     

Quantitative determination of nucleic acids in brown and white adipose tissue

The low-molecular-weight soybean antiprotease C-II is specifically phosphorylated by a cAMP-independent protein kinase (Casein kinase TS) much more readily than casein itself. Unlike the routinely used substrates casein and phosvitin, C-II is not affected either by casein kinases S or by the cAMP-dependent protein kinase. It can be successfully employed therefore for the direct and reliable evaluation of casein kinase TS within preparations still contaminated by other protein kinases. Crude preparations of soybean antiproteases can replace C-II as specifically phosphorylatable substrate of casein kinase TS, though displaying a much lower phosphorylation efficiency.     

A silver staining procedure for nucleic acids in polyacrylamide gels without fixation and pretreatment

Silver staining of nucleic acid has been widely used in molecular marker analysis such as simple sequence repeat (SSR), single-strand conformation polymorphism (SSCP), and amplified fragment-length polymorphism (AFLP). Many alternatives to silver staining methods have been described, but these methods are not efficient or cost-effective. Here we report a silver staining method that requires less than 10 min for one gel and can save chemicals as well. It has a detection limit of approximately 5.6 pg of DNA/mm² in nondenaturing polyacrylamide gels and 12.8 pg/mm² in denaturing polyacrylamide gels.     

A refined method for silver staining of nucleic acids fixed on nitrocellulose

A refined silver staining method was developed to stain nucleic acids fixed onto nitrocellulose membranes and nylon-based membranes. Approximately 4 ng RNA or DNA can be stained with this method with no protein interference. This method involves simple repetition of immersions of membranes in three solutions prepared from common chemicals. The total staining time is less than 30 min.