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PubMed1991

A new stain for pollen fertility studies.

The juice from the berries of Cocculus hirsutum was extracted and used for pollen fertility studies in various crops. Two stains were prepared: P.H. Ramanjini (PHR) stain and modified PHR stain. The modified PHR stain contains lactic acid and produces the best staining differentiation. The intensity of the staining was dependent on the thickness of the pollen cell walls, hence PHR stain is recommended for thick walled pollen grains and the modified PHR stain for pollen with relatively thin walls. The preparation of both the stains are very simple, quick and inexpensive.

Fruit↗

PubMed1992

Quantitative determination of murine dermal elastic fibers by color image analysis: comparison of three staining methods.

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Kligman's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.

Animals↗

PubMed1993

Demonstration of urinary eosinophils in Schistosoma haematobium: a comparative study among three different stains.

Three stains, Hansel's stain, alkaline erythrocin B (AEB) and naphthalene black (NB), were used to demonstrate eosinophils in the urine of patients infected with Schistosoma haematobium. Hansel's stain was superior to the other two stains; it stained eosinophils bright red and their nuclei faint blue, and they were easily differentiated from neutrophils, lymphocytes, macrophages and epithelial cells. The method using AEB took longer than Hansel's stain and 10% of the specimens were lost during staining with this method. Like eosinophils, the neutrophils took up NB stain and their nuclei stained poorly with the counterstain.

Amido Black↗

PubMed1995

Fixation and staining of planaria for histological study.

Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acid-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH.

Animals↗

PubMed2005

Value of endoscopic methylene blue and Lugol's iodine double staining and detection of GST-Pi and telomerase in the early diagnosis of esophageal carcinoma.

AIM: To explore the expressions of GST-Pi and telomerase activity in esophageal carcinoma and premalignant lesions and to investigate the value of endoscopic methylene blue (MB) and Lugol's iodine double staining. METHODS: Seventy-two patients with esophagopathy were sprayed endoscopically with MB and Lugol's iodine in proper order and the areas stained blue and brown, and the area between the blue and brown stains were obtained. Depending on the pattern of mucosal staining, biopsy specimen was obtained. GST-Pi and telomerase activity in specimens were examined by immunohistochemistry and PCR-based silver staining telomeric repeat amplification protocol, respectively. RESULTS: After MB and Lugol's iodine staining, the area between both the colors was obtained in 64 of the 72 patients and the areas were stained blue and brown in all of the 72 patients. Association test of two simultaneous ordinal categorical data showed a correlation between the esophageal mucosal staining and the esophageal histology (P<0.005). The expression of GST-Pi and telomerase activity in esophageal carcinoma and premalignant lesions increased. The expression of GST-Pi and telomerase activity in dysplasia and carcinoma was significantly higher than that in normal epithelium (P<0.005). The expression in hyperplasia was slightly higher than that in normal epithelium. With the lesions progressing from low- to moderate- to high-grade dysplasia, the positive rate increased (P<0.025). Expression of GST-Pi was correlated with that of telomerase activity in dysplasia and carcinoma (phis = 0.4831, P<0.005; phis = 0.3031, P<0.025, respectively); but there was no correlation between them in normal epithelium and hyperplasia. CONCLUSION: The expression of GST-Pi and telomerase may be an early event in the carcinogenesis of esophagus. They may play an induced and synergistic role with each other in the carcinogenesis of esophagus. Endoscopic MB and Lugol's iodine double staining and detection of GST-Pi and telomerase activity may contribute to the early diagnosis of esophageal carcinoma.

Adult↗

PubMed1988

A simple staining method for cryptosporidian oocysts and sporozoites.

A useful staining method for detection of cryptosporidian oocysts and sporozoites was developed and described. The modified Kohn's one-step staining technique with an additional modification, i.e., longer staining time and higher staining temperature than those originally described, was tested on fecal smears from cats infected with Cryptosporidium sp. By this improved staining procedure, the oocyst appeared as a slightly oval body containing four internal sporozoites colored blue to blue-gray. The oocyst wall was stained dark green to black. The morphological feature of the oocyst was recognized much more clearly by this staining technique than by such currently used techniques as acid-fast and Giemsa staining. This staining procedure proved to be simple and less costly and to secure a good preservation.

Animals↗

PubMed2001

Distinctive microvillous brush border staining with HBME-1 distinguishes pleural mesotheliomas from pulmonary adenocarcinomas.

HBME-1 is an antimesothelial monoclonal antibody that recognises an unknown antigen on microvilli of mesothelioma cells. The aim of this study was to evaluate the staining pattern with respect to antibody dilution, cellular distribution and intensity of immunohistochemical staining with HBME-1 in pleural mesotheliomas compared with pulmonary adenocarcinomas. A total of 27 pulmonary adenocarcinomas and 26 mesotheliomas were stained with commercially available HBME-1 at various antibody dilutions and evaluated for the site (membranous, +/- microvillous brush border or cytoplasmic), intensity and percentage of cells staining. On light microscopy, 23 mesotheliomas showed distinctive microvillous brush border staining with HBME-1 (three mesotheliomas--two sarcomatoid and one poorly differentiated--were negative). Twenty-five adenocarcinomas showed membranous +/- cytoplasmic staining but lacked the distinctive microvillous brush border staining. In a subgroup of tumours studied by electron microscopy following immunogold labelling by HBME-1, all of 16 mesothelioma cases showed strong immunogold labelling in the membranes of the long microvilli. In contrast, the 12 cases of pulmonary adenocarcinomas showed minimal labelling in the membranes of the short microvilli, but staining was seen within vesicles, often near the surface of the cells. This study shows that the presence of a distinctive microvillous brush border by immunohistochemical staining with HBME-1 allows distinction between pleural mesotheliomas and pulmonary adenocarcinomas (sensitivity of 88%, specificity of 100%). The difference in the ultrastructural distribution of immunogold labelling with HBME-1 between mesotheliomas and adenocarcinomas underscores the light microscopy findings.

Adenocarcinoma↗

PubMed1996

Uvitex 2B stain for the diagnosis of Isospora belli infections in patients with the acquired immunodeficiency syndrome.

OBJECTIVE: Fluorescent stains with Uvitex 2B or other fluorochromes are widely used today, especially for the diagnosis of microsporidian infections in human immunodeficiency virus (HIV)-infected patients. Our objective was to ascertain whether the fluorescent stain with Uvitex 2B is also able to detect Isospora belli in stool samples and duodenal/bile juice of HIV-infected patients. DESIGN: Case study. SETTING: University hospital of the University of Cologne, Germany. PATIENTS: Two HIV-infected patients with chronic diarrhea who had intestinal infections with I. belli. METHODS: Stool was concentrated by a modified water-ether sedimentation method, and duodenal/bile juice was concentrated by centrifugation. Wet-mount preparations were examined by phase-contrast and bright-field microscopy, and smears were stained with a modified acid-fast stain and a fluorescent stain with Uvitex 2B. RESULTS: Using the fluorescent stain with Uvitex 2B, the oocysts of I. belli stained bright white/blue fluorescent and showed a structure similar to that of the oocysts in acidfast stains. CONCLUSIONS: Staining with Uvitex 2B is a suitable method for the diagnosis of I. belli infections. This technique can be used for the diagnosis of three important gastrointestinal parasites (viz, microsporidia, cryptosporidia, and I. belli) responsible for diarrhea in HIV-infected patients.

AIDS-Related Opportunistic Infections↗

PubMed2003

Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells.

OBJECTIVE: To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells. METHODS: Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient. RESULTS: One hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis. Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50% - 90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or Annexin V-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type. CONCLUSIONS: Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-staining may be used as reference method to clearly identify apoptosis induced by oxidative stress in adherent cells. The TUNEL-reaction, annexin-V-binding and Apo-2.7-expression may be used to quantify the number of apoptotic and necrotic cells especially at later stages but without discrimination of apoptosis and primary or secondary necrosis.

Annexin A5↗

PubMed2003

Enhanced detection of Mycobacteria stained with rhodamine auramine at 37 degrees C.

Sputum smear microscopy is the most efficient and rapid technique for detection of acid-fast bacilli (AFB). Fluorochrome method of staining is preferred for Mycobacteria in the overburdened laboratories as the fluorescing bacilli are more readily detected than the fuchsin stained bacilli in shorter period of time. A total of 300 sputum samples obtained from suspected cases of Tuberculosis were collected and were subjected to staining by rhodamine auramine at 37 degrees C and also at room temperature (conventional method). The smears were then blindly evaluated. Fifty-eight samples were positive by both methods and 5 were positive at 37 degrees C only. Staining at 37 degrees C increased the smear positivity by 8.6% over conventional staining at room temperature. No smears were positive only with staining at room temperature alone. Out of 58 smears positive by both methods, 25 had equal number of AFB in both smears, 22 had more AFB in smear stained at 37 degrees C and 11 had greater number of AFB in smears stained at room temperature. Our study, therefore, indicates that rhodamine auramine staining at 37 degrees C is superior to conventional auramine method at room temperature for detecting AFB in sputum smears.

Benzophenoneidum↗

PubMed2004

[Value of special stains and immunohistochemistry in the diagnosis of renal epithelial neoplasms].

OBJECTIVE: To study the diagnosis and differential diagnosis of renal epithelial neoplasms. METHODS: Ninety-one cases of renal epithelial neoplasms with detailed pathologic records were enrolled. In addition to microscopic examination, Mowy's colloidal iron staining and immunohistochemical studies (CD10, vimentin and CK7) were also performed. RESULTS: Among the 91 cases, there were 78 (86%) clear cell renal carcinoma cases, 8 (9%) papillary renal carcinoma cases, 4 (4%) chromophobe renal carcinoma cases and 1 (1%) renal oncocytoma case. Sixty-three of the 78 clear cell renal carcinoma cases were positive for CD10 and 69 were positive for vimentin (81% and 88% respectively), with prominent cell membrane staining. The majority (74/78) of clear cell renal carcinoma were negative for CK7. All 17 clear cell renal carcinoma cases showed negative or focal coarse droplet-like staining pattern for Mowy's colloidal iron stain. All 4 chromophobe renal cell carcinoma cases showed prominent cell membrane staining for CK7 and blue reticular staining pattern for Mowy's colloidal iron stain. All of which were negative for CD10 and vimentin. The case of renal oncocytoma failed to react with antibodies to CD10, vimentin and CK7, or Mowy's colloidal iron stain. CONCLUSIONS: CD10, vimentin, CK7 and Mowy's colloidal iron stains have proved to be useful in differential diagnosis of common renal tumors which may not be easily distinguished on the basis of histologic examination alone.

Adenocarcinoma↗

PubMed2004

[A comparative study of endoscopic image stained by iodine and histopathology in early esophageal cancer and precancerous lesions (dysplasia)].

OBJECTIVE: The aim of this study is to evaluate the relationship between the imaging patterns of endoscopy aided by iodine stain and histological diagnosis. METHODS: A balloon cytological screening was conducted in 4000 participants in high-risk area of esophageal cancer in 1997-1998, 1050 out of these 4000 participants were confirmed as abnormal by cytology. Among them, 867 were given endoscopy examination during which mucosal stain with 1.2% iodine solution was used. The stain images were graded as four categories: Grade 1, dark yellow, protruding inlay-like with clear borders and showing pink after discoloration; Grade 2, between grade 1 and grade 3; Grade 3, light yellow, flat lesion with clear but not sharp borders and Grade 4, dark-brown color. According to the above criteria, the lesions were recorded and graded after the examination. The biopsies were taken from the unstained lesions and underwent pathologic evaluation. A comparison between the imaging patterns of endoscopy aided by iodine stain and histological diagnosis was evaluated. The correlation coefficient was estimated using Spearman's Rank Correlation. RESULTS: 94.4% (68/72) of the superficial carcinoma and severe dysplasia, 61.8% (73/118) of moderate dysplasia and 27.7% (35/126) of mild dysplasia were distributed in the grade 1 and grade 2 of iodine stain images of which the patterns conforming to the principle and rule of formation of the image by iodine stain. CONCLUSION: There is close relationship between the imaging patterns of endoscopy aided by iodine stain and histological diagnosis. The magnitude of exposure of the malignant tissue is the pathologic basis of formation of various images by iodine stain. Iodine stain greatly helps of early detection of esophageal cancer.

Biopsy↗

PubMed1990

A comparison study of two methods of peanut agglutinin staining with S100 immunostaining in 29 cases of histiocytosis X (Langerhans' cell histiocytosis)

Staining with peanut agglutinin (PNA) and with polyclonal antibody to S100 protein have both been recommended as aids in the diagnosis of histiocytosis X (Langerhans' cell histiocytosis). Although a large body of literature attests to the utility of S100 staining in this condition, the few published studies that involve PNA staining have employed varying methods of radically different results. We studied formaldehyde solution-fixed, paraffin-embedded tissue from 29 cases of histiocytosis X by using standard avidin-biotin immunostaining for S100 protein, as well as two published methods of PNA staining. All cases stained positively for S100 protein. By utilizing prior trypsinization and a three-step procedure, all cases also stained with PNA. With a two-step PNA-staining procedure, however, 9 cases failed to stain with PNA, and 3 cases showed a diffuse staining pattern that was not considered characteristic of Langerhans' and histiocytosis X cells. We concluded that both the S100 and the three-step PNA procedures are sensitive methods for the diagnosis of histiocytosis X.

Adolescent↗

PubMed1991

Planimetry of bronchoalveolar macrophages. Importance of preparation and staining techniques.

Bronchoalveolar lavage seems a well-established, valuable research tool in the study of alveolar macrophages. The influence of fixation, cytocentrifugation and staining procedures on the cellular and nuclear size has been investigated by planimetry. As a reference, mean profile areas of 109 and 39 microns 2 for cell and nucleus, respectively, were measured for alveolar macrophages suspended in the hemocytometer. For comparison, stained Cytospin preparations were measured. Unfixed cells were compressed during cytocentrifugation. The cellular profile areas for Cytospin preparations increased about 15% and 70% after May-Grünwald-Giemsa and Feulgen staining, respectively. The nuclear area was approximately 25% larger for both staining procedures as compared to the hemocytometer values. When the cells had been fixed prior to cytocentrifugation, these differences were less conspicuous. No significant differences were observed after May-Grünwald-Giemsa staining, showing a cellular area of 114 microns 2 and a nuclear area of 45 microns 2. Depending on the staining procedure, low nucleus:cell ratios (31%) were observed after Feulgen staining, while higher values (about 43%) were measured after May-Grünwald-Giemsa staining, regardless of which fixation or centrifugation procedure had been followed. In conclusion, these findings indicate that fixation should be carried out in order to prevent cell changes resulting from cytocentrifugation. Moreover, different staining procedures considerably influence the measurement of cellular and nuclear profile areas and the determination of nucleus:cell ratios.

Analysis of Variance↗

PubMed1985

Amniocentesis for gram stain and culture in preterm premature rupture of the membranes.

A retrospective study was conducted to assess the predictive value and clinical usefulness of amniotic fluid Gram stain and culture in the management of preterm premature rupture of the membranes (PROM). Amniocentesis was attempted in 79 patients and was successful in 53 patients for a success rate of 67%. The complication rate was 1.8%. Fifty-three patients from whom amniotic fluid was obtained after preterm PROM were studied. Patients were managed expectantly unless clinical chorioamnioitis developed, a positive Gram stain or culture was found, or a mature lung profile after 34 weeks' gestation was established. A low incidence of chorioamnionitis (5.5%), postpartum endometritis (11%), and neonatal sepsis (3.5%) was found with expectant management. A statistically significant association between Gram stain and antepartum infection, postpartum infection, and neonatal infection was noted. Gram stain had a 100% sensitivity and 82% specificity as predictor of chorioamnionitis. Amniotic fluid culture had a 100% sensitivity and 76% specificity as predictor of chorioamnionitis. As predictor of a positive culture result, Gram stain had a sensitivity of 60% and specificity of 92%. No patient with a negative Gram stain or culture developed infectious sequelae regardless of the timing of amniocentesis. All positive Gram stains and cultures were found within 48 hours of rupture of membranes. Amniocentesis for Gram stain and culture has clinical usefulness in preterm PROM and, despite false-positive tests, the authors' data support intervention on the basis of a positive Gram stain.

Amniotic Fluid↗

PubMed1985

Reproducibility of morphometric measurements of amyloid after various staining methods.

Four histologic staining methods used for detecting amyloid (Congo red, viewed in both normal and polarized light, Sirius red, Crystal violet and Thioflavine T) were applied to heart muscle autopsy samples from 19 patients who suffered from amyloidosis. The amount of amyloid present was evaluated with morphometry (point counting) by five pathologists, and the interobserver reproducibility and variation of point counting in these staining methods were analyzed. The Sirius red method showed the least variation and was the most suitable stain for demonstrating amyloid with respect to reproducibility. Thioflavine T showed the greatest variation and was the least suitable stain with respect to reproducibility. The range of variation was considerable in all staining methods. The results show that stains differ in their specificity and sensitivity in staining amyloid, observers differ in their interpretation of staining results and certain stains result in more uniform interpretations than do others.

Amyloid↗

PubMedDate not supplied

The congo red stain revisited.

The Congo red stain has undergone several modifications since it was first used by Bennhold in 1922 in order to increase the specificity for staining amyloid. Most of the laboratories in the United States use the method of Puchtler which uses alkaline Congo red solution. Some of the variables associated with the procedure were investigated by us. Our results showed the following: (1) amyloid showed green birefringence at all levels between 4 to 12 mu thick sections with better visualization of small deposits with increased thickness. Best results were obtained with 8 mu thick sections; (2) omission of the pretreatment with alkaline alcoholic solution of sodium chloride (NaCl) did not affect the sensitivity of the method; (3) the use of polar mounting media had no effect on amyloid and collagen birefringence; (4) 50 percent saturation of the Congo red staining solution with NaCl caused strong staining of collagen, elastic fibers and eosinophilic granules. In addition, collagen showed green birefringence and dichroism and its differentiation from amyloid became difficult; and (5) using the staining solution fully saturated with NaCl, no positive staining was seen with tissues other than amyloid. Collagen and elastic fibers showed red fluorescence which was of less intensity than amyloid. It is our conclusion that the method of Puchtler for detecting amyloid gives better results if the staining solution is fully saturated with NaCl. The pretreatment step may be deleted without compromising the quality of staining. Improved staining of amyloid enhances the specificity of green birefringence, dichroism, and red fluorescence.

Amyloid↗

PubMed1976

Staining mycobacteria with carbolfuchsin: properties of solutions prepared with different samples of basic fuchsin.

Acid fast staining of mycobacteria in the form of beadings is obtained by means of a carbolfuchsin solution (Ziehl-Neelsen stain) prepared from pararosaniline or from certain kinds of basic fuchsin. After such acid-fast stains, the intensity of the bacilli's colouring was rather poor and unstable, so that some bacilli lost their acid-fast stain. In contrast, an acid-fast staining of mycobacteria in rod form results by using a carbolfuchsin prepared from rosaniline or from other basic fuchsins included new fuchsin. The spectrophotometric and thin-layer chromatographic data indicate that the main component of those basic fuchsins showing beady staining may be pararosaniline, whereas the main ingredient of basic fuchsin with staining the bacteria in rod form may be its higher homologues. Neither chloride nor acetate of the fuchsin could affect the appearance and number of stained bacilli. The commercially available "basic fuchsin" is either the chloride or acetate of pure pararosaniline or consists of variable mixtures of it with higher homologues. Consequently, only a basic fuchsin which has an absorption maximum at lambda greater than or equal to 552 nm could be employed for the acid-fast stain of mycobacteria in a stable manner. Pararosaniline included some basic fuchsins, composed mainly from pararosaniline, should not be selected for the preparation of the carbolfuchsin formula.

Animals↗