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Factors affecting the formation of metallic silver and the binding of silver ions by tissue components.

The rate of formation of metallic silver has a maximum when plotted as a function of pH. The site of this maximum on a pH scale differs noticeably for various tissue elements. By contrast, the amount of silver ions bound to the tissue is a monotonously increasing function of the pH. A temperature rise decreases the length of the induction period and increases the gradient of the ascending section of the kinetic curve representing the formation of metallic silver. It also increases the maximum amount of silver ions bound to the tissue. An increase in the concentration (activity) of the silver ions in the impregnating bath has the same effect. Chemical composition and concentration of the complexing agent, as well as "special" ions in the impregnating bath to which earlier some definitive role has been attributed in the silver staining methods, proved to be ineffective when both pH and activity of silver ions were kept constant. Illumination of the reaction was also ineffective. The kinetic curves obtained in nonaqueous but polar media (e.g., acetone) exhibited the same qualitative characteristics as those obtained in aqueous solutions. No reaction between silver ions and tissue was observed in apolar solvents.

Histocytochemistry

Kinetics of formation of metallic silver and binding of silver ions by tissue components.

The effect of time on the formation of metallic silver by tissue reducing groups follows a curve which can be divided into three main parts. In the first, which may last for several hours, the reaction is very slow, and only an undetectably small amount of metallic silver is produced. In the second period the speed of the reaction first increases in a progressive manner and then begins to decrease gradually; during the third period the speed approaches zero asymptotically. Binding of the silver ions by the tissue commences initially at its fastest rate; the level then decreases steadily to zero within about a quarter of an hour. There is no direct relationship between the amount of silver ion bound to the tissue and the formation of metallic silver. The latter cannot take place by way of direct (non-catalysed) reaction. The following mechanism is proposed for the process: Transfer of electrons from the reducing molecules to the silver ions is mediated at first by certain tissue sites (catalytic points) and then also by the steadily increasing total surface area of the metallic silver grains (autocatalysis). On the basis of this mechanism, several anomalies of both the argentaffin and argyrophil reactions are explained.

Histocytochemistry

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.

Animals

Simultaneous determination of the amounts of metallic and "reducible" silver in histologic specimens.

Acids and weak complexing agents (pK less than 8) are not able to remove, without leaving a residue, silver bound to biological tissues by ionic or complex bonds ("reducible" silver), whereas, strong complexing agents (pK greater than 8) can also partially or completely dissolve metallic silver formed under the influence of reducing groups in the tissue. For this reason, the chemical nature of the silver contained in tissue sections, be it metallic or reducible, must not be determined on the basis of solubility tests; moreover, the amount of neither of the two above fractions can be determined by removing the other with any kind of washing. Using radioactive impregnating baths, radioactive silver bound to the tissue as reducible silver can be replaced in a quantitative manner with inactive silver ions by means of a one-hour incubation in 1% inactive silver nitrate dissolved in 10% acetic acid, but the radioactive silver existing in reduced (atomic) state will be left unaffected. Consequently, radioactivity remaining in the tissue after the above treatment represents metallic silver. The amount of reducible silver can be calculated by subtracting that of the metallic silver from the total silver content of the sections.

Histocytochemistry

Solubility studies of silver sulfonamides.

The solubilities of silver sulfapyridine, silver sulfamethazine, and silver sulfamethizole as a function of pH were determined in nitric acid-potassium nitrate, acetate, and sulfonic acid buffers. All silver sulfonamides showed an increase in solubility with increasing hydrogen-ion concentration, a behavior which closely paralleled the protonation of the p-amino function of the sulfonamide. A silver-ion selective electrode was used to measure silver-ion concentration in solution and the methods of known subtraction and known addition were used to measure total silver. Both silver sulfamethizole and silver sulfamethazine were ionized completely in solution. Silver sulfapyridine was ionized completely only in the more acidic pH 2-3 range. A comparison of the physical properties of the silver salts for which mortality studies were available revealed a unique set of properties for silver sulfadiazine.

Hydrogen-Ion Concentration

Age-dependent variability of ribosomal RNA-gene activity in man as determined from frequencies of silver staining nucleolus organizing regions on metaphase chromosomes of lymphocytes and fibroblasts.

Frequencies of silver staining nucleolus organizing regions (NORs) have been determined in lymphocytes and fibroblasts from very young and from aged persons. Since silver staining of NORs is associated with activity of ribosomal RNA-genes, we used this approach to investigate a possible inactivation of these genes during aging. Our lymphocyte data are based on a study per age-group of 220 metaphases from 10 subjects. Although in both age-groups modal numbers of silver staining chromosomes per metaphase had similar ranges over the subjects, the frequency of metaphases containing the maximal number of staining chromosomes was in the old age-group (80--89 years) significantly lower than in the young age-group (less than 1 year old). In fibroblasts, of which 75 metaphases from 4 subjects were included per age-group, differences were more pronounced. Modal numbers of silver staining chromosomes were for the aged persons (69--83 years) lower than for the young children (less than 1 year old). Highly significant differences were observed between both groups in frequency of metaphases containing the maximal number of positively reacting acrocentric chromosomes and, more in general, in frequencies of silver staining D- and G-group chromosomes, the lower frequencies being found in the old age-group. We propose the term NOR-junctions as distinct from satellite associations for arrangements of acrocentric chromosomes which after silver staining are visibly connected at their NORs. The number of acrocentric chromosomes involved in lymphocyte NOR-junctions of aged people was significantly higher than the number of joined acrocentrics in young children. The frequency of these NOR-junctions themselves, irrespective of the number of chromsomes involved, was higher for aged persons than for young children, although this difference appeared to be statistically not significantly higher than in fibroblasts. Also based on qualitative observations from our study we discuss tcehnical and biological problems of our approach to study cell aging in vivo by means of silver staining of NORs. We conclude that in man, reflected by the difference in frequencies of silver staining NORs between young and aged persons, a rather extensive loss of ribosomal RNA-gene activity may occur during aging.

Aged

Interrelationship of dietary silver with copper in the chick.

Adding 900 p.p.m. silver (as silver nitrate) to a practical diet for chicks significantly depressed growth, increased wet and dry heart weight to body weight ratios and markedly increased mortality during a four-week experimental period. Blood packed cell volume was not affected. Supplementing the diet containing silver with 50 p.p.m. copper prevented cardiac enlargement and mortality, but only partially corrected the growth depression. Glycogen content of the heart was not affected, but aortic elastin content was significantly reduced by silver and restored to normal by supplemental copper. Dietary silver significantly reduced the copper content of blood, spleen, brain, liver, but except for the brain, the level of copper in these tissues was restored to normal by dietary copper supplementation. No significant differences in copper content of kidney tissue were observed among the treatment. Copper content of the excreta was not significantly increased by adding dietary silver, but was greatly increased by adding 50 p.p.m copper to the diet containing silver.

Administration, Oral

Cytological and histochemical studies on the mechanism of the selective silver staining of nucleolus organizer regions (NORs).

A new silver staining method is presented (Ag-II staining) providing a rapid and reproducible way to selective silver staining of nucleolus organizer regions (NORs). In comparison with other techniques, such as the Ag-AS method and the Ag-I method, factors influencing silver stainability are discussed. Histochemical studies on the nature of the NOR-specific silver precipitate were performed either by employing various pretreatments or by inhibiting the participation ("blocking") of the various proteins or protein compounds in the staining reaction. The results would seem to indicate that the interactions of silver-ions with the carboxyl groups of acidic proteins which are involved in the rRNA-transcription process are mainly responsible for the selective silver staining of NORs.

Animals

Solubility studies of silver sulfadiazine.

The solubility of silver sulfadiazine as a function of pH was determined in nitric acid-potassium nitrate buffer for pH 2-3 and in 2-(N-morpholino)ethanesulfonic acid buffer for pH 6-7. As the salt of a weak organic acid, silver sulfadiazine exhibits the anticipated increase in solubility with an increasing hydrogen-ion concentration. Measurement of the silver-ion concentration was carried out using a silver-ion selective electrode. The methods of known subtraction and known addition were utilized to measure the total concentration of the silver ion in solution. Evidence was obtained to indicate that the salt is completely ionized in aqueous solution.

Chemical Phenomena

Inhibition and killing of oral bacteria by silver ions generated with low intensity direct current.

Silver cations generated by passing low intensity direct current through pure silver electrodes were found to be sufficiently antibacterial to cause sterilization of samples of infected dentin. The optimal procedure involved a 5 microA current applied for 20 minutes with the anode then left in contact with the sample. Minimal inhibitory concentrations of electrically generated silver ions for representative oral bacteria were essentially equal to those for silver ions added as nitrate or fluoride salts, and medium constituents, including sodium thioglycolate, antagonized antibacterial action. A major advantage to the use of the electrode method is that it allows for continuous, focal application of antibacterial silver cations.

Bacteria

Silver deposition in mouse glomeruli.

Administration of 6 mM silver nitrate in the drinking water of mice resulted in deposition within the glomerular basement membrane of silver granules which were detected on electron microscopy after 12 days. Larger aggregates were detected in the basement membrane and mesangium when silver ingestion was extended to 14 weeks. The silver deposits did not alter significantly over a period of 21 weeks after silver nitrate ingestion was stopped.

Animals

An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes.

A reliable technique for staining human chromosomal nucleolar organizers (NOR's) with silver solutions is described. The NOR's can be selectively stained dark brown by silver solutions leaving the chromosome arms unstained and available for counterstaining with orcein or Giemsa dyes. Unequivocal identification of chromosome pairs bearing NOR's can be achieved using fluorescent banding techniques followed by silver staining. The silver staining procedure for NOR's was simplified and standardized through control of the chemical and physical conditions during silver impregnation and developing.

Chromosomes

Pattern of activity of nucleolus organizers during spermatogenesis in mammals as analyzed by silver-staining.

Silver-staining in the nuclei and chromosomes of spermatogenesis of four species of mammals (Man, Mus musculus, Rattus norvegicus, and Cavia cobaya) was investigated qualitatively and quantitatively. These species show a very similar pattern of activity of the nucleolus organizer regions (NORs) during the various stages of spermatogenesis. Silver precipitates are detectable in growing spermatogonia and up until the pachytene stage of meiotic prophase. During the meiotic metaphases I and II and during interkinesis silver-stainability disappears completely. A resumpton of silver-stainability occurs in round spermatids indicating a postmeiotic reactivation of NORs. This process does not persist beyond the early elongation phase. The quantitative determination of the silver-covered areas in relation to the total nuclear areas reveals minor differences between the species investigated with regard to the times and extents of maximum activation. The known localizations of the NORs in the karyotypes of the species investigated was confirmed using metaphase-preparations derived from somatic tissues.

Animals

Rapid identification of chromosomes carrying silver-stained nucleolus-organizing regions: application to case of 21/21 Robertsonian translocation.

The use of a combination of transmitted light and epiluminescence after silver and fluorescent staining of chromosome preparations makes it possible to achieve simultaneous visualization of silver-stained NORs and flourescent chromosomes. This technique permits exact localization of silver precipitates on normal and BrdU-substituted chromosomes. After previous silver impregnation, fluorescent staining by actinomycin-daunomycin-DAPI was used to induce a banding pattern that enables identification of specific chromosomes while observing silver-stained NORs at the same time. Application of this method to a Down's syndrome patient revealed a 21/21 Robertsonian translocation with NOR'S eliminated.

Cell Nucleolus

Visualization of ribosomal gene activity: silver stains proteins associated with rRNA transcribed from oocyte chromosomes.

Cricket oocyte chromosomes were stained with silver at pachytene when certain chromosome regions are active in rDNA amplification and rRNA transcription. The silver preferentially stained the known locations of 18S + 28S ribosomal cistrons. Cytochemical tests revealed that the silver binds neither to the rDNA nor transcribed rRNA, but rather to proteins which rapidly associate with the freshly-transcribed rRNA. As rRNA transcription proceeds, the quantity of silver stainable proteins progressively increases. The silver procedure can be used to visualize gene activity at the rDNA sites with conventional light microscopy.

Animals

Silver staining of myelin by means of physical development.

For staining myelin with silver a physical development technique has been devised that can render visible the thinnest fibers in various animal species, including fishes and reptiles, even in the early phase of myelination and may be applied to both frozen and embedded materials. Its principle is as follows: Myelin can form and bind colloidal silver particles in a 0.1% ammoniacal silver nitrate solution of pH 7.5. The production of metallic silver by other tissue elements is suppressed by the sections pretreated with a 2:1 mixture of pyridine and acetic anhydride for 30 min. The colloidal silver particles bound in the myelin are enlarged to microscopic dimensions by a special physical developer.

Animals

Identification of nucleolus organizer regions (NORs) in normal and neoplastic human cells by the silver-staining technique.

Silver nitrate has been used to demonstrate the chromosomal location of ribosomal cistrons in nine tissue-culture lines derived from human tumors of various pathological origins. Control individuals have a particular modal number (range 7--10) of D- and G-group chromosomes stained with silver. In the controls, 96.2% of the D- and G-group chromosomes that have a stalk show silver staining, while no relationship can be seen in acrocentric chromosomes without stalks. The tumor cells, whose modal chromosome numbers range from 42 to 68, possess variable numbers of acrocentrics (11--18). The number of chromosomes stained with silver, however, remained at control levels (range, 6--9). These data indicate that, in humans, silver staining may not identify all NORs that contain structural ribosomal genes.

Cell Line