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PubMed1975

Contamination of anti-immunoglobulin reagents with antibodies to beta 2-microglobulin and other unrelated antigens. Effects on immunofluorescence staining of human lymphocytes.

IgG fractions from three of four rabbit antisera to Bence Jones proteins of chi-type were found to contain antibodies to beta 2-microglobulin and to stain 80%-100% of human blood lymphocytes by indirect immunofluorescence. Antibody fractions from these sera, which contained anti-beta 2-microglobulin but not anti-Ig, stained all lymphocytes, whereas the isolated anti-Ig antibodies (anti-chi) stained only a minor cell population. In both instances, the specificity of the staining was confirmed by absorption experiments. One antiserum to the constant half of lambda-type Bence Jones protein also contained antibiodies to beta 2-microglobulin and stained all lymphocytes. Four other anti-lambda reagents contained no antibodies to beta 2-microglobulin and stained at most about half of the lymphocytes. The antigen responsible for this staining is unknown. The isolated anti-immunoglobulin antibodies (anti-lambda) stained only 5%-10% of the lymphocytes. Antisera to serum IgG or its fragments were free of antibodies to beta 2-microglobulin and stained only 10%-25% of the lymphocytes. This staining was in all instances due to antibodies to human immunoglobulin. Five of eight undiluted sera from normal rabbits with no detectable antibodies to human immunoglobulin or beta 2-microglobulin stained 25%-60% of the lymphocytes. This staining rapidly disappeared on dilution.

Antibodies↗

PubMed1979

Postmortal "vital" staining of the external eye.

Two hundred and two eyes from 112 individuals were vital-stained from 2 to 46 h after death. Intensity and extension of staining were studied in ten regions. The staining gradually progressed after death, also within non-exposed areas. It most often started anteriorly on the tarsus and finanally included the fornix. Microscopy revealed diffuse cell staining by rose bengal or trypan blue. The cell nucleus was most often more intensely stained than the cytoplasm. Up to five h after death, neutral red had only stained vacuoles in the cytoplasm. Later diffuse staining of cells occurred. Tetrazolium differed from the above dyes in that the pronounced staining seen immediately after death gradually decreased in the course of time. Microscopy disclosed stained inclusion bodies in the cytoplasm up to 18 h after death. Then cell staining was only seen as a rare exception in relation to extracellular dye granules. However, the mucous thread in the inferior fornix showed gradually increasing postmortal tetrazolium staining. The amount of mucus was found to be the same in dead persons as in the living. The characteristic appearance of the dead eye is due, among other things, to ruptures of the corneal epithelium (fluorescein-stained) and cell death, and not to drying up or coating by mucus.

Adult↗

PubMed1990

Evaluation of an indirect fluorescent-antibody stain for detection of Pneumocystis carinii in respiratory specimens.

Two prospective studies were undertaken to evaluate a commercial indirect fluorescent-antibody (IFA) stain for the detection of Pneumocystis carinii in respiratory specimens from individuals at risk for or with the acquired immunodeficiency syndrome. The first study compared IFA with Diff-Quik (DQ; a rapid Giemsa-like stain) for detecting P. carinii in 95 induced sputa obtained from 77 asymptomatic patients who had survived one previous episode of P. carinii pneumonia and who were being treated prophylactically with aerosolized pentamidine. Only one induced sputum specimen was found to contain P. carinii; organisms were detected by both stains. The second study compared the performance of the IFA stain versus DQ, modified toluidine blue O, and Gomori methenamine silver stains for detecting P. carinii in symptomatic individuals at risk for or with acquired immunodeficiency syndrome. Of 182 specimens examined, P. carinii was detected in 105 by one or more stains; the DQ stain detected 73 (70%), the modified toluidine blue O stain detected 75 (71%), the Gomori methenamine silver stain detected 76 (72%), and the IFA stain detected 95 (90%). The IFA stain was more sensitive (P less than 0.01) than the other traditional stains for detecting P. carinii; however, a subsequent clinical evaluation revealed that a subset of IFA-positive-only specimens were from patients whose clinical symptoms resolved without specific anti-P. carinii therapy.

Diagnostic Errors↗

PubMedDate not supplied

Routine air drying of all smears prepared during fine needle aspiration and intraoperative cytology studies. An opportunity to practice a unified protocol offering the flexibility of choosing a variety of staining methods.

OBJECTIVE: To evaluate the possibility of routine use of air-dried smears (ADS) instead of wet-fixed smears (WFS). STUDY DESIGN: Intraoperative cytology (IC) smears from 293 specimens and fine needle aspiration cytology (FNAC) smears from 118 cases were studied. Cytomorphology of ADS processed with our protocol for hematoxylin-eosin (HE) and Papanicolaou (PAP) staining after saline rehydration and postfixation in 95% ethanol with 5% acetic acid were compared with respectively stained WFS. Additional ADS were stored up to 72 hours at room temperature prior to HE, PAP and Diff-Quik (DQ) staining to evaluate the effects of postponing rehydration and postfixation. Special stains for fungi were also studied in four cases. RESULTS: ADS were easy to prepare without air-drying artifact in the final HE- and PAP-stained smears. ADS were more cellular than WFS. Erythrocyte interference was frequent in WFS. HE and PAP staining of ADS stored up to 72 hours showed cytomorphology comparable to that of the similarly stained fresh smears. However, DQ staining was better if ADS were processed before 24 hours. ADS stained with special stain for fungi showed good morphology, similar to that in WFS. CONCLUSION: All ADS showed results comparable to or better than WFS. ADS could be stored up to 72 hours before staining with HE and PAP. ADS offers the flexibility of selecting a variety of staining methods and is a practical alternative to WFS.

Adult↗

PubMedDate not supplied

Staining and in vitro toxicity of dithizone with canine, porcine, and bovine islets.

Dithizone (DTZ) is a recognized diabetogenic agent in vivo, and a supravital stain commonly used for identification of islets to be used for transplantation. In the present studies, we compared DTZ staining of freshly isolated and cultured canine, bovine, and porcine islets, and the effect of DTZ on the function and viability of islets. Incubation with DTZ resulted in staining of canine and porcine islets, but no discernible staining with bovine islets. Insulin content of porcine, canine, and bovine islet was 2.0 +/- 0.2, 2.2 +/- 0.3, and 1.9 +/- 0.2 mU/EIN, indicating a lack of correspondence of DTZ staining and insulin content. Seven days of culture with canine islets resulted in > or = 50% reduction of DTZ stained cells. Exposure to DTZ at 50 micrograms/mL resulted in a maximal number of stained cells in preparations of purified islets (80-85%; counted after dispersion), a lower percentage of cells stained faintly at 20 micrograms/mL (50-55%), with no discernible staining at 10 micrograms/mL. Prolonged exposure of islets (4-48 h) to 20 micrograms/mL DTZ led to reduced insulin secretion and islet cell death. Incubation of canine or porcine islets with 100 micrograms/mL of DTZ for 0.5 h resulted in a dramatic loss of viability and diminished insulin secretory function, which was not reversed with continued culture. The concentration dependence of toxic effects paralleled the concentration dependence of cellular staining. The minimally effective staining concentration (20 micrograms/mL) also resulted in a loss of viability. An additional assessment of DTZ toxicity was made using the RIN-38 beta-cell line, which shows no discernible staining with DTZ.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Fc-mediated nonspecific staining of the porcine brain with rabbit antisera in immunocytochemistry is prevented by pre-incubation of the sera with proteins A and G.

Nonspecific staining was detected in immunocytochemical procedures on the porcine hypothalamus with rabbit antisera, irrespective of the antigen specificity of the sera, in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON), and in the vasopressin- and oxytocin-containing nucleus (VON). The present study was designed to test the hypothesis that this staining is mediated by the Fc portion of rabbit immunoglobulins. Rabbit antisera against neuropeptides localized predominantly outside the PVN, SON, and VON were employed in combination with different detection methods. The intensity of the nonspecific staining varied depending on the antiserum and persisted after pre-absorption of the antisera with their homologous peptides. Nonspecific staining and antigen-specific staining were differentially affected by the method of tissue fixation. The nonspecific staining could be prevented by preincubation of the antisera with proteins A and G, which left the antigen-specific staining intact, whereas additional preabsorption with homologous peptide abolished all staining. These observations suggest that the Fc region of IgGs is indeed involved in the nonspecific staining. On press-blots of homogenates from SON tissue subjected to isoelectric focusing, one band in the low-pH region was found with all antisera. Pre-incubation of the antisera with protein A abolished the staining of this band but did not affect staining of antigen-specific bands. Pre-incubation with proteins A and G is proposed as a routine control to check for nonspecific staining mediated by the Fc region of IgGs in immunocytochemical procedures, particularly those that employ rabbit sera in porcine brain.

Animals↗

PubMed1999

[Development of slide-method to distinguish alive and dead mycobacteria by fluorescent staining--a trial for solving the biohazard problem in TB laboratories].

An acid-fast staining can detect mycobacteria in clinical specimens rapidly and specifically. It equally stains living and dead bacteria. It would be of more clinical use if the viability of mycobacteria in a sample was determined by the staining. In the present paper, the problems of FDA/EB staining, which detects live or dead bacteria, were solved by establishing a new technique, a slide-method. An air-dried smear of Mycobacterium bovis BCG (Tokyo 172) on a glass slide was covered by a filter paper fully soaked in the staining solution (500 micrograms FDA and 40 micrograms EB per ml PBS). This was kept in an incubator at 37 degrees C for 20 min. The filter paper was removed after incubation and the slide was examined using a fluorescent microscope with a blue filter. Live bacteria were stained greenish yellow taking the FDA stain in while dead bacteria were stained red with EB. This new slide technique eliminated the problems associated with FDA/EB staining. Moreover, stained smears appeared to be more stable compared with the conventional tube method. To overcome the biohazard problems in smear examination of tubercle bacilli, heating of the slides on a heat block at 100 degrees C for 20 min or passing air dried smears in a flame 5 to 30 times was tried to kill the bacteria. The heat-treated slides were stained with FDA/EB and the number of green and red bacteria were counted. Samples of the smeared bacteria were taken after heating and cultured on a solid medium to determine the presence of any colony-forming unit. It was found that no CFU was observed after heating and the morphology of the stained sample was the same to that before heating. These facts suggest that the above mentioned method is a simple, safe yet inexpensive diagnostic tool for mycobacterial clinical specimens.

Bacteriological Techniques↗

PubMed2001

Quantification of histochemical staining by color deconvolution.

OBJECTIVE: To develop a flexible method of separation and quantification of immunohistochemical staining by means of color image analysis. STUDY DESIGN: An algorithm was developed to deconvolve the color information acquired with red-green-blue (RGB) cameras and to calculate the contribution of each of the applied stains based on stain-specific RGB absorption. The algorithm was tested using different combinations of diaminobenzidine, hematoxylin and eosin at different staining levels. RESULTS: Quantification of the different stains was not significantly influenced by the combination of multiple stains in a single sample. The color deconvolution algorithm resulted in comparable quantification independent of the stain combinations as long as the histochemical procedures did not influence the amount of stain in the sample due to bleaching because of stain solubility and saturation of staining was prevented. CONCLUSION: This image analysis algorithm provides a robust and flexible method for objective immunohistochemical analysis of samples stained with up to three different stains using a laboratory microscope, standard RGB camera setup and the public domain program NIH Image.

Algorithms↗

PubMed2002

[Study on the method for demonstrating dust granules in phagocytes with Warthin-Starry special staining].

OBJECTIVE: To search a method for showing dust granules in phagocytes. METHODS: The autopsy samples of 10 cases who died of non-pulmonary diseases(group A), 6 cases with silicosis(group B) and 5 cases of dead fetus(group C) were collected. The samples of lung, spleen and lymphnodes were stained by methods of (1)HE staining; (2)Warthin-Starry staining(W-S staining) and modified W-S staining; (3) CD68 (as a first antibody) immunohistochemical staining. The ultrastructure and chemical component of the dust granules in the pulmonary phagocytes(dust cells) of group A(3 cases) and group B(3 cases) were observed and analyzed by transmission electron microscope(TEM) and analytic electron microscope(AEM). RESULTS: In group A and B smaller or minute dust granules which could not be shown by HE staining method were clearly shown by W-S staining and modified W-S staining methods. There were no positive granules found in group C. The CD68 marker of the dust cells was positive. The size, shape, density and chemical elements of the dust granules of group A were different from those of group B under TEM and AEM, excluding bacteria and other intracellular contents. CONCLUSION: The dust granules in the phagocytes could be shown by W-S staining method, its staining effect for minute dust granules is superior to general HE staining. However, the determination of chemical elements of dusts must depend on AEM.

Antigens, CD↗

PubMed2004

[Application of methylene blue staining in detection of Malassezia].

OBJECTIVE: To assess the value of methylene blue and parker ink staining in the detection of Malassezia. METHODS: Detecting Malassezia in three dermatoses samples (pityriasis versicolor, Malassezia folliculitis and dandruff) stained by methylene blue (3 g/L) or parker ink. Comparing the effectiveness and the positive rates of Malassezia detection between the two staining methods. Staining Malassezia and Candida and comparing the specificity of the two staining methods in staining Malassezia. RESULTS: Dark blue hyphae and spores were observed in smears stained by methylene blue, and light blue hyphae and spores were observed in smears stained by parker ink. Hyphae and spores were easy to identify on the background. The positive rates of methylene blue and parker ink for detecting Malassezia in the three dermatoses were 96% and 94% respectively. There was no significant difference between the two dye liquors in the positive rate for deterting Malassezia. Both Malassezia and Candida could be stained by methylene blue; nevertheless, only Malassezia could be stained by parker ink. CONCLUSION: Both methylene blue and parker ink staining can be used to detect Malassezia, but parker ink has higher specificity in Malassezia staining.

Dermatomycoses↗

PubMed2004

Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain.

OBJECTIVE: To find a staining method for formalin-fixed, paraffin-embedded tissue that would distinguish bone from surrounding soft tissues, including muscle, periosteal tissue and bone marrow. STUDY DESIGN: A variety of stains were tested and compared with hematoxylin-eosin. The potential value of any given stain was evaluated based on its ability to stain bone and soft tissues different colors or shades that could be readily identified in photomicrographs. Stains were evaluated using both endochondral (tibia) and intramembranous bone (calvaria) samples. RESULTS: In contrast to standard hematoxylin-eosin stain, which stains both bone and soft tissues pink, the methylene blue/acid fuchsin stain demonstrates remarkable contrast between bone and other tissues. Methylene blue/acid fuchsin stained bone bright pink and the surrounding soft tissues blue-purple. CONCLUSION: In addition to the superior staining properties of methylene blue/acid fuchsin, other benefits of this stain include its stability, ease of use and low cost. This stain has many potential applications in the study of erosive bone disease in humans and also in animal models for research.

Animals↗

PubMed2006

[Comparison of three staining methods for hepatitis B virus].

OBJECTIVE: To compare the advantages and disadvantages of three staining methods of HBV. METHODS: Normal Liver tissue and HBV-infected, HCV-infected, or dually infected (HBV and HCV) liver tissues were selected for this study. Formalin-fixed, paraffin-embedded sections (4 microm) were prepared. Each of the liver tissue specimens was detected by three staining methods, including immunohistochemical methods, Shikata's orcein stain and Victoria blue stain,respectively. RESULTS: In the three methods, all of six HBV-infected cases showed intense staining, and three cases with dual infection (HBV and HCV) were weakly positive. However, both normal and HCV-infected liver tissues showed no staining. HBsAg stained dark brown with Immunohistochemical stain; HBsAg containing ground-glass hepatocytes stained magenta with Shikata's orcein stain; HBsAg stained blue with Victoria blue. CONCLUSION: Each of three methods has its own advantages and disadvantages: high specificity and sensitivity, but high cost for immunohistochemical methods; complicated and overelaborate procedure for preparation of solutions, lower specificity and sensitivity, but low cost, for special staining methods.

Hepatitis B↗

PubMed1991

A comparative study of quantitative stains for DNA in image cytometry.

In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine-chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide.

Analysis of Variance↗

PubMed1984

Staining tissue-derived Mycobacterium leprae with fluorescein diacetate and ethidium bromide.

A fluorescent staining procedure incorporating the use of fluorescein diacetate (FDA) and ethidium bromide (EB) has previously been shown to accurately measure the viability of saprophytic mycobacterial cells. Green-stained cells were shown to be viable and red-stained cells, dead. Staining Mycobacterium leprae cells with FDA/EB, however, was complicated by interfering tissue components which masked the presence of stained bacteria. A petroleum ether separation technique enables M. leprae to be segregated from armadillo liver tissue components and permitted M. leprae to be stained qualitatively equal to the saprophytic mycobacteria. An alternative and technically simpler method of staining M. leprae from human skin biopsies and mouse foot pads was developed which permitted the initiation of a clinical assessment of the staining method. Preliminary data indicate that patients who have undergone three or 24 months of chemotherapy possess a significantly lower percentage of green-stained M. leprae in their tissues than untreated patients. This would be expected if the FDA/EB staining method was providing an accurate measure of viability. M. leprae cells obtained from mouse foot pads which were harvested 5-13 months post-infection displayed more than 90% green-stained cells. There was no correlation between the FDA/EB staining method and the morphological index.

Animals↗

PubMed1994

Utility of Gomori methenamine silver stains in bronchoalveolar lavage specimens.

Bronchoalveolar lavage (BAL) with Gomori methenamine silver (GMS) stain is commonly used to detect Pneumocystis carinii and fungal organisms as causes of infectious pulmonic disease in immunosuppressed patients. However, several reports have indicated that GMS stains are not any more sensitive than conventional cytologic stains in detecting Pneumocystis organisms in select patient populations, such as those with acquired immunodeficiency syndrome (AIDS). To examine the utility of GMS stains in our laboratory, we retrospectively reviewed 243 BALs from 188 patients. Sensitivity of the GMS stain for Pneumocystis and for fungi detection was 100%. Sensitivity for Pneumocystis and for fungi detection by Papanicolaou stain alone was 79% and 88%, respectively; by Diff-Quik stain alone it was 68% and 88%, respectively; and by combined Papanicolaou and Diff-Quik stains it was 79% and 100%, respectively. In four additional cases, fungi were detected by other methods (culture, biopsy) and not by BAL. The GMS stain result was correlated with a number of risk variables to determine which variables were associated with GMS positivity. Using stepwise logistic regression, Pneumocystis positivity by GMS stain correlated (P < 0.0001) only with the variable of history of AIDS or AIDS risk factors. Fungal organism positivity by GMS stain correlated (P = 0.02) only with the variable of history of BAL positivity for fungus. Cost savings analyses were performed, estimating the cost of the GMS stain at $45 (total cost of GMS in 243 BALs was $10,935).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1995

Corneal epithelial fluorescein staining.

BACKGROUND: There is some difference of opinion in the literature about the nature of fluorescein staining of the epithelial surface. Most authors support the view that fluorescein staining is due to drop out of cells and pooling of fluorescein in the footprint. Others believe that fluorescein fills intercellular spaces. Others suggest that cells themselves stain with fluorescein. METHODS: Rabbit corneas were stained with fluorescein and examined with the biomicroscope and later with a higher magnification epifluorescent microscope following excision. RESULTS: Fluorescein staining was shown to be due to staining of individual cells. No evidence was found to support the contention that fluorescein resides in areas of cell drop out, or that staining was due to filling of intercellular spaces. Micropunctate staining is shown to be due to staining of cells in an optimum manner, which is referred to as hyperfluorescence. CONCLUSIONS: The recognition that fluorescein stains cells, even in the case of epithelial abrasion, allows reinterpretation of many staining phenomena, including "salt and pepper" staining.

Animals↗

PubMed1998

Identification of Cyclospora cayetanensis in stool using different stains.

Cyclospora cayetanensis, a newly emerging coccidian protozoa is world-wide in distribution. In the present study, different concentrations and staining techniques were used for identification of Cyclospora. Formol-ether sedimentation and Sheather's sugar flotation were used as concentration techniques and the different stains used were: the modified Ziehl-Neelsen, Giemsa, safranin-methylene blue, modifications of trichrome stain, calcoflour white and finally phenol-auramine. The safranin stain was the best, as it stained all the oocysts of Cyclospora uniformly, besides being rapid and easily applicable in the laboratories. Phenol-auramine stained the oocysts well, where both the wall and internal contents fluoresced brightly. With the calcoflour white stain, only the wall of oocysts took that fluorescent stain. The modified Ziehl-Neelsen stained some of the oocysts well, yet great variability in the staining pattern was noticed. Cyclospora oocysts were not efficiently stained with either trichrome modifications or Giemsa stains.

Animals↗

PubMed1998

Detection of Microsporidia by different staining techniques.

Previous detection of Microsporidia relied mainly on electron microscopy and histopathology. Recently, non invasive methods were able to recognize this microorganism. In the present study, different stains were used as a means of diagnosing spores of Microsporidia in stool samples of immunosuppressed patients. The original modified trichrome stain (MTS) was used as a standard screening technique for all stool samples. Positive samples for Microsporidia were then stained with the trichrome blue stain, Didier's trichrome blue stain, acid-fast trichrome stain (AFT), modified Ziehl-Neelsen stain, giemsa stain and calcofluor white M2R stain. Both calcofluor and the AFT stains were most efficient. They could simultaneously detect coccidial oocysts and microsporidial spores. This is beneficial and time-saving in the diagnosis of stool samples of immunosuppressed patients, which usually contain more than one opportunistic protozoon. Both stains are easy to perform and require the least amount of staining and examination.

Animals↗