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PubMed1985

Comparison of the Scott Selecticult-HSV kit with conventional culture and direct immunoperoxidase staining for detection of herpes simplex virus in cultures of clinical specimens.

In a comparative study, clinical specimens were cultured for herpes simplex virus (HSV). The presence of virus was noted by the appearance of characteristic cytopathic effect, as determined by standard direct immunofluorescence techniques, by using a direct immunoperoxidase stain for viral antigen, or by using the Selecticult-HSV (SC-HSV) stain for viral antigen. There was 100% correlation between the SC-HSV stain and immunofluorescence staining in recognizing HSV-infected cells (81 of 81 positive specimens). In comparison with observation of cytopathic effect, the SC-HSV system and conventional culture detected 93 and 78% of positive cultures at 48 h postinoculation and 76 and 32%, respectively, at 24 h. By 5 days postinoculation, SC-HSV detected 100% of the positive specimens. As compared with the direct immunoperoxidase stain, SC-HSV stain was slightly more sensitive and gave less background stain. HSV serotypes 1 and 2 were both detected by the SC-HSV stain. The Scott SC-HSV kit appears to be an effective system for the diagnosis of HSV infections.

Antigens, Viral↗

PubMed1991

Comparison of monoclonal antibody and calcofluor white stains for the detection of Pneumocystis carinii from respiratory specimens.

Three monoclonal antibody staining kits, from Genetic Systems, Disease Detection International, and Meridian Diagnostics, were compared with calcofluor white for the direct detection of Pneumocystis carinii in respiratory specimens. Of the 150 specimens tested, 23 were found positive for P. carinii by any of the four stains; 13 were bronchoalveolar lavage, 7 were induced sputum and 3 were expectorated sputum specimens. All stains detected the positive bronchoalveolar lavage specimens, the Genetic Systems stain detected six induced sputum specimens, and the other stains each detected four induced sputum specimens. The monoclonal antibody stains detected all three expectorated specimens, while the calcofluor stain detected only one. Overall, the sensitivities of the stains were 96% for Genetic Systems, 87% for both Disease Detection International and Meridian Diagnostics, and 78% for calcofluor.

Antibodies, Fungal↗

PubMed1988

Demonstration of aluminium in iliac bone: correlation between aluminon and solochrome azurine staining techniques with data on flameless absorption spectrophotometry.

The presence of excess aluminium was investigated in 204 samples of iliac bone from 197 patients with chronic renal failure by using the aluminon and solochrome azurine staining techniques. The results were compared with values obtained by atomic absorption spectrophotometry (AAS). Overall, the staining reactions correlated with the AAS data, but the solochrome azurine stain was positive more often than was the aluminon stain (in 90.6% and 62.3%, respectively, of bone samples with greater than the control group mean +3SD (that is, in the range 17.8 to 113.4 micrograms aluminium/g Solochrome azurine staining was consistently positive in all cases, with greater than 23.1 micrograms aluminium/g bone but the corresponding aluminon stain was occasionally inexplicably negative in this range. In some samples solochrome azurine was positive in parts of old unresorbed cement lines when the aluminon stain was negative and the bone aluminium concentration was within the normal range or slightly increased. The stains, particularly solochrome azurine, have the advantage over AAS in that they indicate the location as well as the presence of excess bone aluminium.

Aluminum↗

PubMed1988

Evaluation of faecal preservation and staining methods in the diagnosis of acute amoebiasis and giardiasis.

The use of a faecal preservative and several staining methods, together with formalin ether concentration, were evaluated for the improved diagnosis of intestinal amoebiasis and giardiasis in 1285 patients with diarrhoea or dysentery and from asymptomatic controls. All samples were screened by three wet mount techniques. Thirty eight specimens of diarrhoeal or dysenteric stool were preserved in polyvinyl alcohol (PVA) and stained by trichrome and Spencer and Monroe short iron haematoxylin stain. Thirty nine preserved faecal samples submitted for routine screening were subjected to formalin ether concentration, wet mount examination, and permanent staining. Saline and buffered methylene blue (BMB) mounts were equally good for detection of trophozoite Entamoebae while Giardia trophozoites were detected only by the saline mount. The iodine mount was superior to the other mounts for protozoan cyst detection. The concentration procedure enhanced cyst recovery. Faecal preservation and subsequent staining was superior to wet mount examination for detection of the trophozoite stage and avoided the need for fresh specimens. Both the trichrome and the iron haematoxylin stains were comparable for the detection of cysts and trophozoites of the Entomoebae. Giardia lamblia trophozoites stained better with iron haematoxylin than with the trichrome. Preservation and permanent staining is recommended as the most productive means for the accurate identification of the various protozoan parasites.

Animals↗

PubMed1992

Two-colour immunoenzymatic technique using sequential staining by APAAP to evaluate two cell antigens.

AIMS: To extend the alkaline phosphatase-antialkaline phosphatase (APAAP) immunoenzyme single stain method to a more generally applicable double stain technique. This will allow two primary antibodies of the same isotype of IgG and specifically the nuclear antigen bromodeoxyuridine (BRdU) to be evaluated with a cell surface antigen identifier. METHOD: Sequential applications of the APAAP method showed two antigen sites by different dye couplings to a common alkaline phosphatase substrate, producing blue and red reaction products on the same slide. Antigens on different cell populations as well as those in different compartments of the same cell were analysed. The method allowed a surface antigen monoclonal to be revealed first, using an optimal fixative, before alcohol/gluteraldehyde fixation was used to start the second (BRdU) staining sequence. RESULTS: An analysis of double staining of T lymphocyte subsets (CD4 and CD8) showed no significant difference in the order of application of the primaries (n = 10) and no significant difference from their corresponding single stain results (n = 50), confirming the validity of the technique where antigens are exclusively distributed. Other examples, including antigens distributed in different compartments of the same cell, displayed discrete staining which implied validity. CONCLUSION: Double staining by APAAP with this technique seems to be applicable to those cases where antigens are exclusively distributed and includes cases where different compartments of the same cell are stained. It is especially useful in revealing antigens that require different fixation and preparation--that is DNA incorporated BRdU with a surface antigen. But it does seem to have a limited ability to produce a dual colour at a common site.

Antibodies, Monoclonal↗

PubMed1994

Non-specific fluorescent whitener stains in the rapid recognition of pulmonary dirofilariasis: a report of 20 cases.

BACKGROUND: Solitary lung nodules in humans caused by the dog parasite Dirofilaria immitis are steadily increasing in number. The organisms can be easily missed in haematoxylin and eosin stained tissue when they are degenerated and pale staining. METHODS: The value of Tinopal CBS-X (TCBS-X) and Calcofluor white (CFW), two rapid, inexpensive, simple non-specific fluorescent whitening stains, were assessed in the identification of these worms. Deparaffinised rehydrated tissue slides prepared from the pulmonary nodules were stained for one minute in 1% w/v aqueous solutions of TCBS-X or CFW, counterstained, coverslipped, and viewed with a fluorescent microscope. RESULTS: The stains demonstrated the intact worm and worm fragments in 20 cases of pulmonary dirofilariasis collected from hospitals in Houston. The filariae and fragments of filariae stained bright green while the background tissue stained red, delineating the internal structures of the worm. CONCLUSIONS: Dirofilariasis should be included in the differential diagnosis of subpleural masses, and non-specific fluorescent whitening stains can help in the rapid recognition of the fragmented organism in cytological or surgical material.

Adult↗

PubMedDate not supplied

Cytologic diagnosis of Chlamydia in cervicovaginal secretions. Use of a Papanicolaou stain modification with buffered wright solution.

OBJECTIVE: To carry out a prospective study on 1,569 females to evaluate the diagnostic utility of adding buffered Wright solution to Papanicolaou stain for observing cytoplasmic inclusions in cervicovaginal cytology. STUDY DESIGN: Group A had multivacuolated cells and group B, granules (not in a vacuole) in the cytoplasm of low-intermediate epithelial or parabasal cells. There were 7 patients in group A and 16 in group B. Two duplicate plates of cervicovaginal secretions were obtained from each patient before and after treatment; one was stained with Papanicolaou stain and the other with our variant. The trial of therapy consisted of doxycycline (effectiveness for statistical analysis = 0.9) in oral doses of 100 mg every 12 hours for 7 days; 7 days after the beginning of treatment, check samples were obtained. RESULTS: In group A, six patients had cytoplasmic inclusions with the variant stain, and two had cytoplasmic inclusions with Papanicolaou stain. The binomial test revealed that probably six of the seven patients had infections with Chlamydia (P = .372) and that in 100% of cases, the variant stain showed cytoplasmic inclusions, while Papanicolaou stain was observed in 33% of cases. These findings were morphologically and statistically proven (P = .124) on control slides with posttreatment absence of multivacuolated cells. CONCLUSION: In relation to the selection criteria for B group, the intracytoplasmic granules found in parabasal and low intermediate cells had no relation to Chlamydia. The study demonstrated the superiority of our variant of Papanicolaou stain for cervicovaginal Chlamydia diagnosis.

Buffers↗

PubMed2000

Value of gram stain examination of lower respiratory tract secretions for early diagnosis of nosocomial pneumonia.

Except for bronchoalveolar lavage, the value of the Gram stain examination of respiratory tract samples for the diagnosis of hospital-acquired pneumonia (HAP) and their potential impact on empiric antibiotic treatment have rarely been assessed. During a 14-mo period, both plugged telescoping catheter (PTC) and endotracheal aspirate (EA) were performed when an HAP was suspected in mechanically ventilated patients. The results of Gram stain examinations and cultures and previous and subsequent antibiotic treatment were prospectively recorded. Two criteria for pneumonia were considered: (1) clinically diagnosed pneumonia, according to attending physicians, based on clinical and radiological evolution and the PTC culture results, and (2) microbiologically proven pneumonia (solely based on the result of quantitative PTC culture). Of 91 episodes of suspected HAP in 51 patients, 27 (30%) episodes were clinically diagnosed as pneumonia. When considering clinically diagnosed pneumonia, the sensitivity and specificity of Gram stain examination were similar (respectively, 89% and 62% for EA and 67% and 95% for PTC). When considering microbiologically proven pneumonia, the sensitivity and specificity of Gram stain examination were, respectively, 91% and 64% for EA and 70% and 96% for PTC. The negative predictive value of Gram stain examination of EA and the positive predictive value of Gram stain examination of PTC were high. Our results suggest that the combination of Gram stain examination of paired PTC and EA may contribute to the early diagnosis of HAP in about two-thirds of mechanically ventilated patients, and guide the empiric therapy when needed. In the remaining one-third of patients, the Gram stain examination is not helpful in predicting HAP.

Bacteria↗

PubMed2002

Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)

Animals↗

PubMed1994

Canine mast cell tumors: a comparison of staining techniques.

Twelve histochemical methods; affinity staining with avidin peroxidase, wheat germ agglutinin, and concavalin-A agglutinin; and an immunohistochemical stain with Kp1 (CD68) antibody were compared for their relative effectiveness in staining canine mast cell tumors. Stains were compared in 28 mast cell tumors and 19 histiocytomas. The effectiveness of the histochemical methods and the lectins decreased as the mast cells became less differentiated. None of the staining methods were positive on histiocytomas. Periodic acid-Schiff (PAS) gave positive results in a few cases of mast cell tumors where other histochemical stains were negative. Although avidin peroxidase and Kp1 antibody stained more mast cell tumors than any other method, they did not differ significantly from Luna's method, toluidine blue pH 0.5, toluidine blue pH 4.5, alcian blue pH 2.5, safranin O, Unna's method, and Giemsa. No stain was ideal for the diagnosis of canine mast cell tumors; however, this study suggests that the use of avidin peroxidase, Kp1 antibody, and PAS may give additional information for individual poorly differentiated tumors without substantial increase in time or cost.

Animals↗

PubMed1979

Staining properties of aldehyde fuchsin analogs.

This investigation was designed to clarify the role of the aldehyde component of aldehyde fuchsin in its staining reactions. Several aldehyde fuchsin analogs were prepared by using different aldehydes. The staining quality of these analogs and pararosaniline-HCl was compared with that of aldehyde fuchsin prepared with paraldehyde in the usual way. The major findings of this investigation include: 1) Aldehyde fuchsin staining of nonoxidized pancreatic B cells requires a stain prepared with either paraldehyde or acetaldehyde. 2) An aldehyde moiety is required for aldehyde fuchsin staining of strong tissue anions. 3) Staining of elastic tissue with aldehyde fuchsin analogs resembles staining of strong tissue anions more than staining of nonoxidized pancreatic B cells. Possible reaction mechanisms of aldehyde fuchsin with tissue substrates are discussed.

Aldehydes↗

PubMed1980

Separation of viable T and B lymphocytes using a cytochemical stain, Hoechst 33342.

Data are presented that show that a histochemical stain, Hoeschst 33342, can be used to discriminate between viable B and T lymphocytes in the mouse. Quantitative analysis of the staining of cells from various lymphoid tissues with Hoechst 33342 using a Fluorescence-Activated Cell Sorter (FACS) indicates that two populations of cells can be identified. In the spleen approximately 60% of the lymphocytes can be classified as brightly stained with 1 microgram/ml of Hoechst 33342, while in the lymph node only 40% of the cells stain brightly. Thymocytes exhibit only the dull staining profile. Separation of these two populations from the spleen using the FACS and reanalyzing them for cell surface antigenic markers shows that the lymphocytes stained brightly with Hoechst 33342 are predominantly immunoglobulin positive, while the cells that stain less brightly express Thy 1.2. This indicates that a histochemical stain correlates directly with classical immunological markers on cell surfaces.

Animals↗

PubMed1982

A stable propidium iodide staining procedure for flow cytometry.

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.

Animals↗

PubMed1984

The effect of Tween 20 on indirect immunoperoxidase staining of blood group antigen A in human urothelium.

The effect of the nonionic detergent Tween 20 on background staining, sensitivity, and specificity in the indirect immunoperoxidase staining for blood group antigen A was investigated histologically and spectrophotometrically. Pretreatment of dewaxed formalin-fixed Paraplast-embedded tissue sections from human ureters with 2% Tween 20 and dilution of the first and second layer antisera with 0.05 or 2% Tween 20 significantly reduced background staining of the urothelial cell cytoplasm, ureteral stroma, and musculature. Spectrophotometrical analysis of tissue sections from hypernephroma (rich in cytoplasm), cervix (fibrous stroma), and myometrium (musculature) underlined the histological results with a significant reduction of the maximum absorbance of Tween 20-modified indirect immunoperoxidase-stained tissue sections. Sensitivity, evaluated histologically by the endpoint titers of urothelial cell membrane staining, endothelial cell staining, and focal cytoplasmic staining of urothelial cells, was not influenced by the Tween 20 treatment. The specificity was improved as the staining was highly reduced or absent in control sections subjected to Tween 20.

ABO Blood-Group System↗

PubMed1988

Effects of alpha-L-fucosidase digestion on lectin staining in human pancreas.

We examined the effects of alpha-L-fucosidase digestion on lectin staining in formalin-fixed, paraffin-embedded human pancreatic tissue from individuals of different blood groups. Digestion with the enzyme resulted in apparent diminished intensity of Ulex europaeus agglutinin-I (UEA-I) staining in the acinar cells. In addition to the decreased intensity of UEA-I staining, reactivity with soybean agglutinin (SBA) was increased in the enzyme-susceptible, UEA-I-reactive cells. The intensity of Griffonia simplicifolia agglutinin-II (GSA-II) staining performed after beta-galactosidase digestion in UEA-I-reactive acinar cells was markedly increased by prior treatment with fucosidase. GSA-II staining following sequential digestion with fucosidase and galactosidase was completely abolished by subsequent digestion with beta-N-acetylhexosaminidase. These results therefore substantiate the previous assumption that SBA-reactive D-galactose-(beta 1-3,4)-N-acetyl-D-glucosamine and GSA-II reactive beta-N-acetyl-D-glucosamine imparted following galactosidase digestion represent precursors of H antigen. The present study further demonstrated that intense peanut agglutinin (PNA) staining was imparted after digestion with fucosidase in UEA-I-reactive sites in secretors. In contrast, nonsecretors showed vivid PNA staining that was usually detected throughout the pancreas without prior enzyme digestion. Here, fucosidase digestion had if any little effect on PNA staining. These results suggest that in secretors a terminal trisaccharide, fucosylated D-galactose-(beta 1-3)-N-acetyl-D-galactosamine exhibiting positive PNA reaction after fucosidase digestion, exists in UEA-I-reactive acinar cells. It is assumed that the secretor gene could control the step of final fucosylation of D-galactose-(beta 1-3)-N-acetyl-D-galactosamine in human pancreas.

Adolescent↗

PubMed1990

Characterization of six textile dyes as fluorescent stains for flow cytometry.

Few fluorescent stains specific for cell constituents other than DNA are available. To assess their potential use as fluorescent stains for flow cytometry, the cell staining specificity of 55 compounds, originally synthesized for use as textile dyes and fluorescent brighteners, was explored and their excitation and emission wavebands determined. From these, six dyes were chosen for more detailed analysis. All six are vital stains, with excitation wavelengths allowing their use with an argon ion laser, and specific for a range of cell structures including mitochondria, Golgi bodies, lipid droplets, nuclear membrane, and endoplasmic reticulum. Concentrations as low as 0.01-0.25 microM were found to be adequate for most purposes, and high background fluorescence was not a problem. Their specificity allows differentiation between non-cycling and cycling cells. The properties of two of the stains allows their combination with propidium iodide or ethidium bromide for simultaneous determination of DNA content profiles. Being vital stains, usable at very low concentrations, and specific for a range of cell organelles, these six stains may be of considerable utility in flow cytofluorometry. We suggest that other textile dyes may be of use in flow cytofluorometry, or that their structures may form a starting point for the synthesis of further fluorescent stains of enhanced specificity.

Animals↗

PubMed1990

Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

Antibodies, Monoclonal↗

PubMed1992

Alternative staining methods for Lowicryl sections.

A number of stains and stain combinations have been identified that, when used with the hydrophilic resin Lowicryl K11M, produce marked improvements over aqueous uranyl and lead salts (UA-Pb) in terms of low granularity, specificity, and range of components contrasted. Three test specimens, tobacco mosaic virus (TMV), starfish sperm, and cultured mouse fibroblasts, were used to evaluate stain characteristics. UA-Pb showed a preference for nuclei acids, which were stained specifically by osmium ammine-B at pH 1.5. A number of stain combinations in which UA was followed or preceded by salts containing barium, manganese, tungsten, molybdenum, and vanadium provided excellent staining of protein-containing components, each stain combination being unique in terms of the degree to which specific components were discriminated. These stains were particularly effective for visualizing internal components of the nucleus where a number of fibrillar and particulate structures not seen with UA-Pb were well contrasted.

Acrylic Resins↗