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PubMedDate not supplied

Azure B-eosin staining of blood cells: the effects of variation in stain formulation and staining technique on stain performance.

A quantitative study of azure B-eosin-stained blood cells is reported. The effects of variation in stain formulation and staining technique on the binding of azure B and eosin by acidophilic, basophilic and neutrophilic substrates were measured by scanning microdensitometry. The variables considered were stain concentration (the azure B-eosin concentration ratio was constant), azure B concentration (at constant eosin concentration), eosin concentration (at constant azure B concentration), staining time, buffer pH, metal salt contamination, dye contamination, buffer concentration and fixation time. The last two were the only variables which failed to produce changes in dye uptake. Tentative explanations of the observed effects are advanced.

Azure Stains↗
PubMed1997

The staining of acidic proteins on polyacrylamide gels: enhanced sensitivity and stability of "Stains-all" staining in combination with silver nitrate.

A number of acidic proteins, such as those found in bone and dentin, are poorly resolved on acrylamide gels using Coomassie blue or silver nitrate staining. The cationic dye Stains-all allows visualization and identification of these proteins due to their differential staining: highly acidic proteins stain blue and intact proteoglycans stain purple, whereas less acidic proteins stain pink. However, the use of Stains-all is limited due to relatively poor staining sensitivity and lack of stability to light. A procedure which addresses these deficiencies has been developed utilizing established protocols for Stains-all staining followed by silver nitrate incubation and development. In this way, phosphoproteins such as osteopontin, bone sialoprotein, dentin phosphophoryn, and other acidic glycoproteins are visualized at higher sensitivity (greater than fivefold) and staining stability than normally achieved with just Stains-all. The protocol stains a greater variety of proteins than a combined alcian blue/silver staining procedure previously described. Utilizing the Stains-all/silver protocol, porcine bone osteopontin, a protein not visualized by standard silver staining, can be observed in amounts as little as 0.25 ng on polyacrylamide gels. Furthermore, densitometric scans demonstrate that the staining intensity is proportional to osteopontin amount and can be used for quantification over a range from 0.25 to 50 ng.

Acrylic Resins↗
PubMed1987

Standardization of the Romanowsky-Giemsa stain: the influence of staining time on the RG-staining pattern.

In this paper the influence of staining time on the staining pattern of Romanowsky-Giemsa (RG) type stains is investigated. Smears of rabbit bone marrow and of human venous blood were stained with azure B-eosin Y, azure A-eosin Y and with the cationic dyes alone under varying conditions of staining time and dye concentration. The stained smears were investigated by integrating microdensitometry. DNA-polyacrylamide (PAA) model films were stained with azure A-eosin Y, the extinction of the stained model films was determined by spectrophotometry. With increasing staining time the color of the cell nuclei changed from blue to an intense purple, the texture of the nuclear chromatin became more prominent. Prolonged staining resulted in over-staining of the cell nuclei with loss of a distinct chromatin texture. Besides such factors as dye concentration and pH of the staining solution standardization of staining time may be considered necessary for the reproducibility of the RG staining pattern.

Animals↗
PubMed1998

The modified Steiner stain: a new use for an old stain? Staining cytomegalovirus-infected cells in gastrointestinal biopsies.

The modified Steiner stain is a non-specific silver stain for identifying bacteria in formalin-fixed, paraffin-embedded tissues. The principle behind its use is that bacteria are first sensitized using uranyl nitrate solution, making them able to precipitate silver from a silver nitrate solution. It is used routinely for staining gastric biopsies to identify Helicobacter pylori. Upon staining a gastric biopsy from a patient with acquired immunodeficiency syndrome (AIDS) and cytomegalovirus gastritis, we recognized that this technique also stains the viral inclusions of cytomegalovirus-infected cells. We then proceeded to stain 43 consecutive cytomegalovirus-positive gastrointestinal biopsies from 33 immunocompromised patients based on positive cytomegalovirus immunohistochemistry (DAKO-cytomegalovirus monoclonal antibody, clones DDG9 and CCH2). We also stained eight cytomegalovirus-infected, non-gastrointestinal tissues, including lung, adrenal gland, ovary, skin and neural tissue, to ensure that the stain was staining the cytomegalovirus-infected cells and not argyrophilic or argentaffin neuroendocrine cells of the gastrointestinal tract. In 40 of the 43 cytomegalovirus-infected gastrointestinal biopsies, we saw positive staining with the modified Steiner stain (93% sensitivity). The cytomegalovirus-infected, non-gastrointestinal tissues all stained positively with the modified Steiner stain. Because the modified Steiner stain is frequently used to identify Helicobacter pylori in gastric biopsies, we propose that it be studied further for possible use either as a screen or as a confirmatory tool, or both, for cytomegalovirus inclusions in gastrointestinal biopsies.

Cell Nucleus↗
PubMed1995

Double staining of coomassie blue-stained polyacrylamide gels by imidazole-sodium dodecyl sulfate-zinc reverse staining: sensitive detection of coomassie blue-undetected proteins.

The sensitivity, simplicity, and relative rapidity of Coomassie blue staining have made this technique the method of choice for routine detection and quantitative analysis of gel electrophoresis-separated protein bands in many applications. To extend the usefulness of this technique, we have developed a new double-staining method for visualizing SDS-PAGE-separated protein bands that were undetected by Coomassie blue staining of the gel. Coomassie blue-stained gels are washed in distilled water (15 min, two times) and then subjected to imidazole-zinc reverse staining. As a result of the method, a homogeneous white-stained background is generated and two types of protein bands can be observed: (a) typical Coomassie blue-stained bands, which appear superposed on larger transparent bands; and (b) reverse-stained (transparent) bands, which were previously undetected by the Coomassie blue staining. The method is rapid, simple, and reproducible and double-staining gels can be kept in distilled water for months without loss of the protein pattern. The overall sensitivity is high (e.g., 1.6 ng for recombinant streptokinase, 47 kDa) over a wide range of protein molecular weights (10 to 100 kDa) and independent of the degree of Coomassie blue destaining of the gel. Furthermore, a mechanism offering a consistent explanation for the role of imidazole, SDS, and zinc in the reverse staining of gels, particularly after Coomassie blue staining is proposed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Staining sections of water-miscible resins. 1. Effects of the molecular size of stain, and of resin cross-linking, on the staining of glycol methacrylate embedded tissues.

Penetration of hydrophilic acid and basic dyes into sections cut from glycol methacrylate (GMA)-embedded tissues was studied; as were the effects on such staining of superficial coatings of thin layers of GMA. Dye size was a major factor in controlling penetration of resin and staining of tissues. 'Large' dyes (greater than 1000 Da) entered GMA very slowly, and only stained those tissue components poorly infiltrated by resin. 'Small' dyes (less than 550 Da) penetrated GMA readily, and stained tissue components whether or not they were resin-infiltrated. Dyes of intermediate size penetrated the resin, but the staining of resin-infiltrated tissue elements was slow. Background staining of resin also varied with dye size. Large dyes gave no staining of GMA. Small dyes did, but were readily removed by water washing. Dye of intermediate size penetrated resin slowly, and once inside were lost slowly. This gave background staining which required use of the plasticizing solvent ethanol for its removal. Increases in resin cross-linking also reduced staining rates. As a consequence, it is possible to predict the probable suitability, or otherwise, of various staining reagents proposed for use with GMA sections; and also the probable influences of histoprocessing on stain penetration. In particular it is suggested that penetration of colloidal metals and macromolecular reagents (e.g. labelled antibodies and lectins) will be limited to resin-free structures, and to the surface of resin sections. The use of superficial GMA coatings as convenient semipermeable membranes for enzyme histochemistry is also noted.

Animals↗
PubMed1992

Staining sections of water-miscible resins. 2. Effects of staining-reagent lipophilicity on the staining of glycol-methacrylate-embedded tissues.

Glycol methacrylate (GMA) sections of animal tissues were stained with a group of twenty-seven reagents of very varied chemical characteristics. The artefactual background staining of the resin was found to be dependent on the hydrophilic/lipophilic character of the staining reagent, as estimated from the logarithm of its octanol-water partition coefficient (log P). Intense background staining occurred with lipophilic stains, whose log P greater than 2. In keeping with this, use of GMA semi-permeable membranes for enzyme histochemistry failed to give staining when using a lipophilic substrate, probably because the substrate was trapped in the membrane. An analysis of other routine histochemical stains--in terms of the probable occurrence of high resin background staining and low tissue sensitivity--is made. A numerical guide is provided to help avoid artefacts resulting from hydrophobic and size effects. Note: small, hydrophilic reagents (log P less than 0; molecular weight less than 550 Da) are least likely to show either type of artefact. Conversely, reagents which are lipophilic, or/and of intermediate size (log P greater than 2; 550 less than ionic weight less than 1000 Da), give strong background staining.

Animals↗
PubMed1987

Microwave-stimulated staining of plastic embedded bone marrow sections with the Romanowsky-Giemsa stain: improved staining patterns.

Staining plastic sections with the Romanowsky-Giemsa method is both time-consuming and difficult. This paper reports how the staining time can be reduced to 25 min using microwave irradiation of the staining solution. It is shown that staining results depend on the fixative used, staining temperature, dye concentration and pH of the staining solution as well as on several parameters of the microwave irradiation technique. The staining patterns are improved when compared with those obtained by conventional staining of plastic sections. The colors are more brilliant and greater contrasts are observed. Basophilia, polychromasia, and orthochromasia accompanying red cell maturation are more pronounced. For white cell maturation the initial appearance of specific granules (neutrophil, basophil, and eosinophil) is more evident. Thus, cell classification is easily accomplished using the described technique. It is suggested that microwave-stimulated staining be considered for routine use.

Animals↗
PubMed1981

Structure-staining relationships in histochemistry and biological staining. Part 3. Some comments on the intentional and artifactual staining of lipids.

The choice, basis and calculation of certain numerical coefficients for describing chemical structures of such staining reagents as dyes, enzyme substrates and visualising agents as described. The general value of electric charge, conjugated bond number, Hansch pi values, and molecular or ionic weights is emphasised. Hansch pi values as indicators of hydrophobic character, are then used to analyse reagents giving rise to the staining of neutral lipids. Dyestuffs used to stain lipids all have high Hansch pi values (greater than or equal to +1.0), with superior stains, e.g. Sudan Black B and Oil Red O, having values of greater than or equal to +7.0. In keeping with this, conversion of the basic dyes Nile Blue and Brilliant Cresyl Blue, with Hansch pi values of -2.4 and -3.6, into their oxazone derivatives, with Hansch pi values of +4.4, and 3.6, generates lipid staining compounds. Also in keeping with this correlation of lipophilia with Hansch pi values greater than +1.0 is the occurrence of artifactual lipid staining in enzyme histochemistry. Such artifacts can arise for instance when using certain naphthyl substrates which give rise to naphthoic intermediate reaction products, or when tetrazolium salts as used as visualising agents, yielding formazans as final reaction products. The Hansch pi values of the naphthols and formazans generated histochemically typically fall into the lipophilic range. Another artifact of lipid staining is the staining of basophilic entities, such as cell nuclei, by fat stains which carry amino substituents. Calculation of the Hansch pi values for protonated (and hence cationic) derivatives of such dyes yields values typical of basic dyes.

Animals↗
PubMed1992

Refractile mycobacteria in Romanowsky-stained bone marrow smears. A comparison of acid-fast-stained tissue sections and Romanowsky-stained smears.

The appearance of mycobacteria was studied in Wright-stained bone marrow preparations of human immunodeficiency virus-infected patients and compared with acid-fast-stained trephine biopsy sections and culture results. Mycobacterium avium complex in Romanowsky-stained preparations may be seen as extracellular and intracellular clear or red refractile beaded rods and nonrefractile "negative images." Refractile mycobacteria were seen in 17 of 20 culture-positive cases. Acid-fast stain of the trephine biopsy demonstrated organisms in only 11 of the 20 cases. Thus, six cases were culture positive and contained refractile rods but had no acid-fast organisms on the trephine biopsy. No false-positive results were seen with Romanowsky stain; the three false-negative results for refractility also were negative with acid-fast stain. Examination of Romanowsky-stained smears or imprints for refractile mycobacteria provides a reliable and sensitive method to identify mycobacteria in this population. Romanowsky-stained bone marrow aspirate and imprint smears should be examined for refractile bacilli when mycobacterial infection is suspected.

Acquired Immunodeficiency Syndrome↗
PubMed1996

A comparison of sputum examination for acid fast bacilli by modified Schaeffer & Fulton stain, Ziehl-Neelsen stain & cold stain.

A comparative study of Ziehl-Neelsen stain, cold stain and modified Schaeffer and Fulton stain was carried out to evaluate the efficiency of modified Schaeffer and Fulton method in sputum examination for acid fast bacilli (AFB). Of 187 sputum samples studied, 67 (35.82%) were reported positive by Ziehl-Neelsen stain and cold stain method while 66 (35.29%) were reported positive by modified Schaeffer and Fulton method. In comparison with Ziehl-Neelsen staining, 98.58 per cent positivity was reported by modified Schaeffer and Fulton method. Modified Schaeffer and Fulton method is found to be simple, reliable, less expensive and as efficient as Ziehl-Neelsen stain and cold stain for demonstration of acid fast bacilli in sputum.

Bacillus↗
PubMed1980

Structure-staining relationships in histochemistry and biological staining. I. Theoretical background and a general account of correlation of histochemical staining with the chemical structure of the reagents used.

A description is given of the choice, rationale and calculation of several numerical coefficients for describing the chemical structures of staining reagents such as dyestuffs, enzyme substrates, diazonium and tetrazolium salts. Using such coefficients, structure-staining correlations may be made for many staining systems. Examples include the structural correlations of the quantitative affinity measurements of the staining of chromatin with basic dyes, the tetrazolium salts for use as visualizing agents in enzyme histochemistry, and the use of bis-azo compounds as acid or basic dyes, or as fat stains. The value of such structure-staining correlation models for assisting the rational selection and design of new histochemical reagents, and for understanding old reagents and procedures, is discussed.

Chemical Phenomena↗
PubMed1980

Structure-staining relationships in histochemistry and biological staining. II. Mechanistic and practical aspects of the staining of elastic fibres.

Correlations between the structural features of dyes and staining performance for elastic fibres were investigated. Dyes studied included the traditional stains (such as Gomori's Aldehyde-Fuchsin and Weigert's Resorcin-Fuchsin), acid dyes used from alkaline aqueous-organic solvent mixtures (the Horobin-James system), and basic dyes used from acidic aqueous-ethanolic mixtures (the Taenzer-Unna system). In all three classes effective elastic fibre stains had large conjugated bond numbers, and were often hydrophobic (i.e. had high Hansch pi values). By choosing dyes with conjugated bond numbers at or over a critical value (25 for the TU system, 35 for the HJ) it is possible to select new and effective dyes for use in the HJ and TU staining systems. Mechanistically these results support the view that for typical commercial dyes and also for the traditional stains van der Waals attractions provide the important contributions to dye-elastic fibre affinities, with hydrophobic bonding playing a subsidiary role. However, supporting the views of Lillie, it was also noted that even hydrophilic dyes of low conjugated bond number could stain elastic fibres, if the dye carried a sufficiently reactive primary amino group as a substituent. The additional substituent groupings needed to generate such reactivity have been specified, for both acidic and alkaline reaction conditions.

Animals↗
PubMed2001

Comparison of Chemicon SimulFluor direct fluorescent antibody staining with cell culture and shell vial direct immunoperoxidase staining for detection of herpes simplex virus and with cytospin direct immunofluorescence staining for detection of varicella-zoster virus.

A new rapid direct immunofluorescence assay, the SimulFluor direct fluorescent-antibody (DFA) assay, which can simultaneously detect herpes simplex virus types 1 and 2 (HSV-1 and -2) and varicella-zoster virus (VZV), was evaluated in comparison with our current standard procedures of (i) shell vial direct immunoperoxidase (shell vial IP) staining and cell culture for detection of HSV and (ii) cytospin DFA staining for VZV detection. A total of 517 vesicular, oral, genital, and skin lesion specimens were tested by all three procedures. For HSV detection, the SimulFluor DFA assay had an overall sensitivity, specificity, positive predictive value, and negative predictive value of 80.0, 98.3, 92.3, and 95.1%, respectively, when compared to culture. Shell vial IP staining had a sensitivity, specificity, positive predictive value, and negative predictive value of 87.6, 100, 100, and 96.9%, respectively, when compared with cell culture. The SimulFluor DFA assay, however, offers same-day, 1.5-hours results versus a 1- to 2-day wait for shell vial IP staining results and a 1- to 6-day wait for culture results for HSV. For VZV detection SimulFluor DFA staining detected 27 positive specimens as compared to 31 by our standard cytospin DFA technique--a correlation of 87.1%. A positive SimulFluor reaction for VZV is indicated by yellow-gold fluorescence compared to the bright apple-green fluorescence observed by cytospin DFA staining. There is no difference in turnaround time between the two assays. The SimulFluor DFA assay is a rapid immunofluorescence assay that can detect 80% of the HSV-positive specimens and 87% of the VZV-positive specimens with a 1.5-h turnaround time.

Animals↗
PubMed1987

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods.

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.

Animals↗
PubMed1978

Cross contamination of cytological smears, with automated staining machines and bulk manual staining procedures. With a specific study of the problems of the Cytotek and the Shandon Elliott staining machines.

Further development of an individual staining machine is to be strongly encouraged but meanwhile, using bulk stainers, frequent changing of wash fluids and staining solutions, particularly leading up to and following the haematoxylin pot, is essential to reduce the risk of cross contamination. Certain smears, such as from semen or from serous fluids where malignancy is suspected or known, must be stained on separate racks. In some laboratories it is the rule not to stain semen or serous fluids in bulk staining machines at all and this may have to become the rule everywhere until we are provided with safe individual slide stainers.

Adenocarcinoma↗