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PubMed1985

A rapid method of staining ultrathin sections for surgical pathology TEM with the use of the microwave oven.

A rapid microwave method is described for staining ultrathin sections for surgical pathology transmission electron microscopy (TEM). Three sets of Epon sections of human heart biopsy were mounted on unsupported 200-mesh Rhodium-coated copper grids and were stained with uranyl acetate (UA) and lead citrate. The first set of grids was stained for 15 seconds in each solution with the aid of a microwave oven, and the second set was stained routinely for 30 minutes in UA and 10 minutes in lead citrate at room temperature. The third control set was stained for 15 seconds in each solution without microwave bombardment. The overall image quality of the TEM micrographs generated by the "quick-stained" microwave enhanced sections was better than routine stained and control sections. The microwave-treated sections have more contrast, less artifacts in the form of precipitate, and a more uniform overall staining.

Humans↗

PubMed1988

Basic blue 93. An alternative to the traditional myeloperoxidase stain.

This report describes the selective staining of lysosomes in cells of neutrophilic granulocytic origin by the azo textile dye basic blue 93. Used as a single agent aqueous stain after fixation, the dye stained lysosomes black. Stained in this way, lysosomes were sharply delimited and easily visualized. Large numbers of black-stained lysosomes occurred in immature granulocytes like promyelocytes and myelocytes. In more mature granulocytes like metamyelocytes, bands, and neutrophils, substantially fewer numbers of lysosomes were detected. In leukemic myeloblasts and promyelocytes stained with basic blue 93, variable numbers of black-colored lysosomes could be detected and usually paralleled the reactions for myeloperoxidase and Sudan black B in the same cells. As an alternative to the traditional stains for myeloperoxidase, basic blue 93 has several advantages, including simplicity of use, ability to stain aged specimens, and stability of the reaction product.

Azo Compounds↗

PubMed1989

Visual detection of microencapsulated insecticides with selective staining and scanning electron microscopy.

Selective staining with Sudan IV and methylene blue for light microscopy and scanning electron microscopy (SEM) were investigated to determine their potential for detecting and quantifying microencapsulated insecticides. Penncap-M (microencapsulated methyl parathion), Penncapthrin (microencapsulated permethrin), and Dyfonate (microencapsulated fonofos) were selectively stained with Sudan IV but not with methylene blue. Selective staining was not possible for Altosid SF-10 or SR-20 (microencapsulated methoprene) with either stain. Sudan IV enabled detection of some microencapsulated formulations in the digestive content of selected aquatic invertebrates and prepared contaminated pollen samples. Staining intensity with Sudan IV was greatest with acetone but capsular damage was high. A solvent ratio of 50:50 and 20:80 acetone/xylene minimized capsular collapse and maintained good staining intensity. The use of SEM for capsule identification and quantification depended upon the method of sample preparation: the slide smear method was superior to samples prepared by incision or microtomy. SEM was most suitable for investigation of formulations such as methoprene, for which selective staining was not possible. The chemical basis of staining with Sudan IV and potential application of both identification techniques are discussed.

Aedes↗

PubMed1990

The influence of staining procedures on the assessment of cell proliferation as defined by the monoclonal antibody Ki-67.

We examined the influence of different staining techniques [(three-step immunoperoxidase technique (IP); alkaline phosphatase-anti-alkaline phosphatase technique (APAAP)] on the quantitative evaluation of Ki-67-labeled nuclei. We studied five melanocytic skin tumors. From each case, five parallel sections were prepared and stained using the peroxidase-antiperoxidase (PAP) technique (slide 1) and the APAAP technique once (slide 2). Slide 3 consisted of a single repetition of the APAAP technique, slide 4 was a double repetition, and slide 5 was a third repetition. We assessed the volume fraction (VV) of Ki-67-positive nuclei using computer-assisted image analysis. For each staining group, the mean value and standard deviation of VV were calculated. Comparing VV values obtained from the different staining groups we did not find a statistically significant difference between the IP and the various APAAP steps (Wilcoxon test, p = less than 0.05). However, the staining procedure influenced the quantitative results to some extent. The mean VV of the five staining groups ranged in our study from 0.10 to 0.17%, which is narrow compared with the overall variability among different cases (dermal melanocytic nevus, 0.01%; metastatic malignant melanoma, 0.43%). Therefore, we can state that for a rough evaluation of Ki-67-positive nuclei, the influence of different staining methods is negligible; for a subtle quantitative analysis, however, it would nevertheless be preferable to always apply the same staining technique.

Antigens, Surface↗

PubMed1983

A comparative study of generic stains for carcinoid secretory granules.

Generic stains for secretory granules in carcinoid tumors often yield inconsistent and unpredictable results. Consequently, these techniques have been modified and new ones devised; however, very few studies comparing the efficacy of the various methods have been published. To determine which method would most consistently demonstrate carcinoid granules, we applied a battery of six stains to 73 tumors, all morphologically identified as carcinoids, from various sites (17 pulmonary, 13 small intestinal, 20 appendiceal, 18 rectal, five miscellaneous). Stains employed included four argyrophil (Sevier-Munger, Grimelius, Pascual, Churukian-Schenk), one argentaffin (Fontana-Masson), and one non-silver technique (lead hematoxylin). Of the 73 carcinoids, 97% stained positively with the Churukian-Schenk, 82% with the Pascual, 79% with the Grimelius, 74% with the Sevier-Munger, 67% with lead hematoxylin, and 51% with the Fontana-Masson technique. The two cases negative by the Churukian-Schenk technique were also negative with all other stains; thus, this technique most consistently demonstrated secretory granules in carcinoids, including all 18 rectal tumors. The Churukian-Schenk method achieves its superior results by reducing background staining and nonspecific precipitation to a level that permits sparse, small granules to be readily identified. In control sections of normal mucosa and in some tumors, this stain revealed more than one population of argyrophil cells as identified by the size, color, and staining intensity of the granules.

Carcinoid Tumor↗

PubMed1992

Corneal staining characteristics after sequential instillations of fluorescein.

Twenty-one healthy noncontact lens-wearing subjects were screened for the presence of corneal staining with fluorescein after sequential instillations of the vital stain (Phase I). These staining characteristics were further explored by monitoring the results of sequential instillations of fluorescein at 2-h intervals over a 12-h period (Phase II) and by patching 1 eye overnight and observing corneal staining after sequential instillations of fluorescein in the morning after patch removal (Phase III). In Phase II more eyes stained in the early morning. Throughout the day the most common staining areas were inferior and nasal. In Phase III, 9 of 12 patched eyes presented with fluorescein staining, as did 7 of the 12 unpatched eyes. Corneal staining after sequential instillations of fluorescein is more likely to be observed early in the day and is not reduced by overnight patching.

Adolescent↗

PubMed1998

Assessment of clinical outcome after flashlamp pumped pulsed dye laser treatment of portwine stains: a comprehensive questionnaire.

A generally accepted method to assess the clinical outcome of laser treatment of portwine stains is not available. This paper describes the development and evaluation of a comprehensive questionnaire for the assessment of the following portwine stain characteristics: color (hue and lightness), sharpness of boundary, pigmentation, size, shape, skin surface-structure, and hypertrophy of underlying tissue. The questionnaire was applied to photographs of 70 patients with previously untreated portwine stains in the head/neck area. Photographs were taken before treatment and after five treatments of the entire portwine stain with a flashlamp pumped pulsed dye laser. Each photograph was evaluated separately by a panel of five professionals: the treating physician, two plastic surgeons, a dermatologist, and a clinical photographer. Treatment results were assessed by comparing ratings before and after treatment. Agreement among raters was acceptable for all portwine stain characteristics, as was shown by weighted kappa analysis. The reliability of the answers was further improved by taking the average rating of the five panel members. The scores thus obtained were very reliable, with Cronbach alpha coefficients of 0.8 on average. After five treatments of the entire portwine stain, the most considerable changes were measured in the ratings for color (lightening of the stain by 33 percent), boundary (sharpness reduced by 38 percent), and size (13-percent decrease). Using the questionnaire can be helpful in the design of comparative clinical trials on portwine stain treatment and may facilitate comparison of treatment results between different treatment centers and/or different lasers.

Adolescent↗

PubMed1987

A simple metachromatic and fluorescent staining method for microorganisms using carbocyanine dye.

The cationic carbocyanine dye, 1-ethyl-2-[3-(1-ethylnaphtho[1, 2d]-thiazolin-2-ylidene)-2-methylpropenyl]-naphtho[1, 2d]thiazolium bromide, interacts with several classes of anionic polymers, exhibiting metachromasia. We were able to stain various kinds of microorganisms with this dye. Gram-negative bacteria were stained reddish purple, while Gram-positive bacteria were stained violet or bluish purple. Stains of molds were of various colors. Yeast vegetative cells were stained reddish purple, but zygotic asci were bluish. Chlamydia trachomatis inclusions, which are surrounded by cytoplasmic membranes, were also stained red. Microorganism and cell stains have different features and can be identified also by use of fluorescent microscopy. The new staining method we report here is rapid and simple enough for routine microscopical examinations of smears of clinical specimens including microorganisms.

Adult↗

PubMed2006

Standardization of sample preparation, staining and sampling methods for automated sperm head morphometry analysis of boar spermatozoa.

The accuracy of computer-assisted sperm morphometry analysis (CASMA) depends on the careful preparation, fixation and staining of spermatozoa. The efficiency of CASMA may be enhanced by developing optimized protocols. The aim of the present study was to evaluate the influence of sperm washing and the use of three staining techniques [rapid Panoptic, Hemacolor and Harris's Haematoxylin (HH)] on image-processing accuracy and boar sperm head morphometry. Sperm washing had a significant effect on samples stained with rapid Panoptic, increasing the percentage of correctly binarized sperm heads and the contrast between cells and background. However, rapid Panoptic yielded the lowest percentage of properly digitized sperm heads. HH provided the highest cell/background contrast, and also greater sperm head staining intensity, but discrimination of sperm midpieces was considered insufficient. Hemacolor occupied an intermediate position, providing acceptable colour intensity and satisfactory cell/background contrast. Use of different staining procedures prompted dimensional differences in sperm head morphometry. Significant differences between animals were observed for all morphometric parameters. Low within-animal variation coefficients reflected a homogeneous sperm head population. Between-animal variation coefficients were relatively high for Hemacolor and HH, and significantly high for the rapid Panoptic stain. Using Panoptic and HH, stable morphometric measurements required at least 100 properly digitized sperm heads rather than 200, while Hemacolor required only 50 spermatozoa. These results indicate that both washing of semen and staining procedures significantly affect the accuracy of image processing and sperm head dimensions. Hemacolor and HH proved to be the best staining techniques for evaluating sperm head dimensions in boar.

Animals↗

PubMed1982

Specificity of stain distribution in electron micrographs of protein molecules contrasted with uranyl acetate.

The mechanism of contrast enhancement of protein molecules by negative staining with uranyl acetate has been investigated by analysing electron micrographs of microcrystals of the human immunoglobulin Dob. Digitally filtered micrographs were compared systematically with idealized reference images which were constructed computationally, starting from knowledge of the primary sequence and three-dimensional crystal structure of this IgG molecule. By separately modelling negative staining as bulk exclusion of heavy metals, and positive staining as the specific decoration of charged amino acid residues, and then combining these simulated images, we were able to assess quantitatively the amount of positive staining present in micrographs of ostensibly 'negatively stained' proteins. At a resolution of 2 nm, we find that the experimental images do indeed exhibit predominantly negative staining, the best matches being obtained by simulations which also include a minor contribution (10-40%) of positive staining. We have also compared two independent measures for the significant resolution present in images of periodic biological specimens: (i) the outermost visible orders of optical diffraction patterns, and (ii) the band-limited resolutions of the idealized simulations when they most closely match the experimental images. These criteria observe close correspondence, thus vindicating the traditional practice of inferring resolution from the optical diffraction spectra of indirectly represented (stained) objects.

Computers↗

PubMed1984

Chromoxane cyanine R. II. Staining of animal tissues by the dye and its iron complexes.

The staining properties of chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) have been studied. Used alone, the dye imparted its red colour to nuclei, cytoplasm and collagen. The dye was extracted by mild alkali but not by acids. Stainability required ionized amino groups in the tissue, and there was also evidence for non-ionic binding of the dye. The colours obtained by staining with mixtures of chromoxane cyanine R and ferric chloride varied with the molar iron:dye ratio and with the pH. Useful staining was seen only between pH 1 and 2. The tissues were coloured either all blue (when Fe:dye was high), or both red and blue (when Fe:dye was low). Lower pH favoured the deposition of red, higher pH the deposition of blue colour. The red was mainly in cytoplasm, blue in nuclei and myelin. Collagen fibres were red or purple, depending on pH and iron:dye ratio. Red colours were differentiated by acid and changed to blue, but not extracted, by mild alkali. The red substance in the stained sections was clearly not the free dye, so it was probably an iron-dye complex. From the effects of various differentiating agents, it was deduced that the red and blue dye-metal complex molecules were bound to the tissue by the dye moiety, not by interposition of iron atoms. Staining by the complexes of iron(III) with chromoxane cyanine R did not involve nucleic acids or other polyanions or the amino groups of proteins. There was evidence for only non-ionic binding of both red and blue complexes. It is suggested that the red colour in sections stained by solutions with low iron:dye ratio is due to a simple carboxylate complex, [ Fe2H (dye)]-. The blue colour would then result from withdrawal of a proton from the red complex to give [Fe2(dye)]2-. The bases that remove the protons may be arginine-rich nucleoproteins of nuclei and phospholipid bases of myelin. Techniques are described for informative simultaneous staining in two colours, and for the selective staining of either nuclei or myelin.

Animals↗

PubMed1997

The medical necessity for treatment of port-wine stains.

BACKGROUND: Port-wine stains are congenital vascular malformations that can be disfiguring and may lead to psychosocial as well as medical complications. The 585-nm pulsed dye laser is very effective in treating port-wine stains. Laser treatment is often viewed by insurance companies as a "cosmetic procedure" and not "medically necessary". Consequently many patients are denied coverage for treatment of their disfiguring birthmarks. OBJECTIVE: To determine variability of insurance coverage for laser treatment of port-wine stains from state to state. Natural history, progression, and potential complications of port-wine stains are reviewed and rationale for consistent insurance coverage for laser treatment of port-wine stains is given. METHODS: A questionnaire was mailed to 40 dermatologic surgeons in 22 states and the District of Columbia. We reviewed the literature regarding port-wine stains and their potential complications, and health care policy guidelines regarding "medical necessity" and "cosmetic procedures". RESULTS: Insurance coverage for laser treatment of port-wine stains varies from state to state. CONCLUSION: Based on current health care policy guidelines, laser treatment of port-wine stains should be regarded, and covered, as a medical necessity by all insurance providers.

Age Factors↗

PubMed1994

The value of amniotic fluid interleukin-6, white blood cell count, and gram stain in the diagnosis of microbial invasion of the amniotic cavity in patients at term.

PROBLEM: Subclinical microbial invasion of the amniotic cavity occurs in 18.8% of women with term labor and intact membranes and in 34% of patients with term PROM and is a risk factor for the development of puerperal infection related morbidity. Although amniotic fluid white blood cell count, interleukin-6 determination, and Gram stain examination have been used for the diagnosis of intrauterine infection in patients with preterm labor and preterm premature rupture of membranes, no information is available about the accuracy and specific cut-off values for these tests in patients at term. The purpose of this study was to compare the performance of the amniotic fluid Gram stain examination, white blood cell count, and interleukin-6 determination in the identification of microbial invasion of the amniotic cavity in patients at term with and without PROM. METHOD: Amniotic fluid was retrieved from 148 patients with term gestations (90 patients with spontaneous labor and intact membranes and 58 patients with PROM). Samples were cultured for bacteria and Mycoplasma species. Amniotic fluid Gram stain, white blood cell count, and interleukin-6 determinations (ELISA, sensitivity: 43 pg/ml) were performed in all samples. Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture for microorganisms. Analysis was conducted using Mann-Whitney U test, Fisher's exact test, receiver operating characteristic curves and logistic regression. RESULTS: Patients with spontaneous labor and intact membranes: The prevalence of microbial invasion of amniotic cavity in this group was 15.6% (14/90). The most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determination (sensitivity for: interleukin-6 > or = 5.7 ng/ml = 86%, white blood cell count > or = 20 cells/mm3 = 64%, Gram stain = 28%). The most specific test was the Gram stain of the amniotic fluid (specificity for: Gram stain = 84%, interleukin-6 = 79% and white blood cell count = 63%). Multiple logistic regression demonstrated that amniotic fluid interleukin-6 concentration was the only covariate that retained statistical significance when intrauterine infection was used as outcome variable. Patients with PROM: The prevalence of a positive amniotic fluid culture in this group was 39.7% (23/58). Logistic regression demonstrated that only interleukin-6 retained a significant relationship with the results of amniotic culture when all variables were entered simultaneously into a model to predict amniotic fluid culture results. The most sensitive tests for the detection of intrauterine infection were interleukin-6 determination and white blood cell count (sensitivity for interleukin-6 > or = 3.4 ng/ml and white blood cell count > or = 20 cells/mm3 = 69.6% for both). The most specific test was Gram stain (97.1%). CONCLUSIONS: Amniotic fluid interleukin-6 determination is the best rapid test for the detection of microbial invasion of the amniotic cavity in patients at term with and without PROM. When this test is not available, amniotic fluid Gram stain and white blood cell count represent valid diagnostic tools to assess the microbial state of amniotic cavity.

Amniotic Fluid↗

PubMed1984

Histological staining as a measure of stress in collagen fibers.

It has been reported previously that collagen fibers will stain either red or green by Masson's and other trichrome methods depending on whether they have been respectively stressed or relaxed prior to fixation. This was shown in skin [1, 2, 3] tendon [4, 5] bone [6] and films of collagen [7]. If this stain-stress dependence is of a unique quantitative nature, then staining could be used as a tension probe for collagen fibers. Relaxed and stressed collagen bundles of rat tail tendon and rat Achilles tendon have been stained using various staining periods, and results indicate that the change in staining may be associated with denser packing of the fibers in the bundle under stress rather than directly due to the stress itself. Denser packing may reduce the rate of penetration of the counterstain thus causing the staining differences. Since this rate of penetration is dependent on a number of other variables (unrelated to stress), it is concluded that collagen staining is not a reliable tension probe.

Achilles Tendon↗

PubMed1997

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.

Ampicillin Resistance↗

PubMed2001

Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold.

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.

Cyanobacteria↗

PubMed1967

Fluorescein-labeled beta-glucosidase as a bacterial stain.

Fluorescein isothiocyanate-labeled beta-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.

Bacillus anthracis↗

PubMed1981

New, special stain for histopathological diagnosis of cryptococcosis.

The Masson-Fontana silver stain for melanin was employed for the differentiation of pathogenic fungal species in human or mouse tissues. The fungi studied were Candida albicans, Candida tropicalis, Candida glabrata (Torulopsis glabrata), Cryptococcus neoformans, Cryptococcus bacillisporus, Coccidioides immitis, Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Rhizopus rhizopodiformis, and Aspergillus fumigatus. The tissue sections stained with Masson-Fontana silver stain showed a dark brown to black color in the wall of cryptococci, whereas the walls of remaining fungal species were hyaline, except for those of S. schenckii. The yeastlike cells of S. schenckii showed faint brown pigment on the wall. Cultures of these fungi showed staining characteristics identical to those of the in vivo results. Cultures of four nonpathogenic Cryptococcus species, Cryptococcus uniguttulatus, Cryptococcus laurentii, Cryptococcus terreus, and Cryptococcus luteolus, were also tested for staining by the Masson-Fontana procedure. Of these, only C. laurentii stained positively, and the pigment on the cell wall was as intense as that of the cells of C. neoformans. These results indicate that the Masson-Fontana silver stain can be used as a specific stain in the histological diagnosis of cryptococcosis.

Adolescent↗