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PubMed2000

Staining of melanocytic neoplasms by melanoma antigen recognized by T cells.

We stained benign melanocytic nevi and malignant melanoma with antibodies to melanoma antigen recognized by T cells (Mart-1) to determine if this was useful in differentiating benign from malignant melanocytic neoplasms. Forty-five primary malignant melanomas and 71 benign melanocytic nevi were stained with antibodies to Mart-1. Two cases of malignant melanoma metastatic to lymph node and three cutaneous metastases of malignant melanoma were also stained. The degree of staining was graded into diffuse positive staining, focal positive staining, and negative staining. Thirty-six of 45 primary malignant melanomas stained diffusely positive with antibodies to Mart-1. This included three of five desmoplastic malignant melanomas that showed positive staining. Four melanomas showed faint or focal positive staining. One of two metastases to lymph node showed strong positive staining and one showed no staining. All three cutaneous metastases showed diffuse positive staining. Sixty-one of 71 melanocytic nevi showed no staining or faint staining with antibodies to Mart-1. Ten of 71 melanocytic nevi showed strong positive staining. The majority of these were congenital nevi. Staining with antibodies to Mart-1 antigen was a useful marker of malignant melanoma. However, staining may also be seen in benign melanocytic neoplasms. The presence or absence of staining for Mart-1 antigen cannot be used to differentiate benign melanocytic nevi from malignant melanocytic tumors.

Antigens, Neoplasm↗

PubMed1994

Staining mast cells for morphometric evaluation on an image analysis system.

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.

Animals↗

PubMed1979

[Simplification of the Papanicolaou stain which is easily reproducable (author's transl)].

A simple and well reproducable modification of the Papanicolaou stain is reported. The most important modification is the replacement of the natural stain hematoxyline by the qualitively excellent synthetic stain thionin which is immediately added to the alcoholic fixation solution for a fixation stain. The high labor intense alternation between alcohol water and alcohol is thus eliminated. The quality of the nuclear staining is very good. The fixation staining with thionin without counter-staining of the cytoplasma may be used for a rapid thionin stain which gives sufficient cytoplasmatic staining by the thionin for a rapid cytological smear for cancer. The counter-stain of the cytoplasma was also simplified. The over-all staining of this simplification of the pap smear is comparable to that of the original papanicolaou stain. This modification of the papanicolaou stain fulfills requirements which a stain in competition with the original papanicolaou stain should have. 1. The modification is qualitively equal to the original method, 2. the staining method is simpler, 3. the staining method is well reproducable, 4. the staining method is more economical.

Female↗

PubMed2002

Diagnostic utility of immunohistochemical staining for p63, a sensitive marker of prostatic basal cells.

Diagnostically reliable identification of prostatic basal cells has depended on staining for high molecular weight cytokeratin. The diagnosis of malignancy is often based on the absence of basal cells. False-negative staining is occasionally observed. Thus, a second method of identifying basal cells might prove useful. Selective expression of p63, a homologue of p53, has been demonstrated in prostatic basal cells. We investigated the diagnostic utility of p63 staining in 70 consecutive specimens for which the differential diagnosis included prostatic adenocarcinoma: 68 needle biopsies and 2 transurethral resection blocks. High molecular weight cytokeratin staining was the gold standard when material was available. A total of 61 specimens were diagnosed as carcinoma, 4 as atrophy, 2 as high-grade prostatic intraepithelial neoplasia, 2 as unclassified collections of benign glands, and 1 as carcinoma versus high-grade prostatic intraepithelial neoplasia. Sections mounted on charged slides were used for p63 staining for 14 specimens. Sections previously hematoxylin and eosin stained on uncharged slides were used for 56 specimens. In every case in which there was successful p63 staining (55 specimens), basal cells in benign lesions were properly marked and other cell types were not stained. Uninformative staining in the remaining 15 specimens was due to failure of tissue adherence in 14 specimens in which sections were on uncharged slides and, in 1 specimen, to poor positive internal control staining of benign glands. Thus, p63 staining was informative in 55 of 56 specimens (98%) for which there was material for examination. No case with satisfactory p63 and high molecular weight staining showed disagreement between the two stains. An additional group of 21 transurethral resection specimens was stained (p63 and high molecular weight cytokeratin). There was less false-negative staining for p63 compared with the case of high molecular weight cytokeratin. No false-positive staining was seen. We conclude that p63 staining is at least as sensitive and specific for the identification of basal cells in diagnostic prostate specimens as is high molecular weight cytokeratin staining.

Adenocarcinoma↗

PubMed2000

The chemical stain removal properties of 'whitening' toothpaste products: studies in vitro.

BACKGROUND: A considerable number of toothpastes are available as tooth whitening products. Most appear to contain ingredients that might remove extrinsic stains rather than change natural tooth colour. Extrinsic stain removal could be achieved by physical or chemical means. AIM: The purpose of this study was to measure the chemical stain removal properties of a range of whitening toothpaste products and experimental formulations using a standardised method in vitro. MATERIALS AND METHOD: 5 separate studies were conducted involving a total of 39 agents of which 28 were whitening products, 7 were experimental formulations, 2 were oxidising mouthrinses used as positive controls, 1 was a popular fluoride toothpaste product as a benchmark control, and 1 was water as the negative control. The formulations and controls varied in each study. The stain model was saliva/chlorhexidine/tea stain developed on optically clear acrylic to an optical density of at least 2.0. Groups of stained specimens were exposed to standard slurries or solutions of each test agent for 1 minute periods up to 5 minutes. Optical density readings were taken at each 1 minute time point. Analyses were based on per cent stain remaining after 5 minutes and time to 75% stain remaining. RESULTS: 3 toothpaste products achieved 100% stain removal by 5 minutes; 2 of these in 3 out of 4 studies in which they were used. 4 experimental formulations also achieved 100% stain removal. In general agents with high total stain removal also had short times to 75% stain remaining. The majority of agents tested had low total chemical stain removal and prolonged times to 75% stain remaining. A few agents were little different from water and several similar in effect to the conventional fluoride toothpaste. This method in vitro tests agents under the best case scenario conditions for chemical stain removal. CONCLUSION: Only a small number of the whitening toothpaste products have good chemical stain removal potential; the majority are unlikely to achieve their claimed benefits through chemical stain removal. There is clearly a need for further data on the actual effects of such products using both methods in vitro and particularly in vivo or in situ.

Acrylic Resins↗

PubMed2006

Narrow-spectrum staining pattern of Pityrosporum.

BACKGROUND: Broad-spectrum fungal stains are used to detect fungal organisms, but narrow-spectrum stains can assist in fungal differential diagnosis. These stains include mucicarmine and Fontana-Masson (FM) for Cryptococcus, Alcian Blue for Cryptococcus and Blastomyces, Congo Red for Blastomyces and Coccidioides, and Ziehl-Neelsen for some examples of Blastomyces and Histoplasma. Pityrosporum is increasingly being recognized as a pathogen capable of significant cutaneous and systemic infections, but the narrow-spectrum staining pattern of Pityrosporum has not yet been systematically studied and reported. METHODS: Paraffin-embedded archival tissue from 10 skin biopsies containing Pityrosporum was stained with Alcian Blue, Congo Red, Ziehl-Neelsen acid-fast stain, and FM stain. In addition to the 60-min staining time, a modified FM stain with 15-, 30-, and 45-min staining times was also performed. RESULTS: All cases stained positive with Alcian Blue, Congo Red, and FM stains and negative with the Ziehl-Neelsen stain. The modified FM stain with the 15-min staining time showed significant staining in more than 50% of the cases. DISCUSSION: The narrow-spectrum staining pattern of Pityrosporum distinguishes it from Cryptococcus, Blastomyces, Candida, Histoplasma, and Coccidioides.

Biopsy↗

PubMed1982

Lead staining in the Golgi complex.

Lead ions at similar concentrations to those used for Gomori type phosphatase localization stain some parts of the vacuolar system, particularly compartments of the Golgi complex (GC) and isolation envelopes (im) in a characteristic way in both vertebrates and invertebrates. After fixation in 2.5% glutaraldehyde, lead citrate in acetate or aspartate buffer (pH 5.5-7.2) leaves the contents of GC cisternal compartments with a fine particulate stippling. In the fat body of Calpodes ethlius and in mouse pancreas the staining is faint but definite without further enhancement of contrast, although it is easily overlooked after section staining. The distribution of lead stain differs from that of the lead phosphate precipitated after Gomori type acid phosphatase reactions. Whereas lead stain may be in all GC and im compartments, acid phosphatase is restricted to the innermost saccules and nearby vacuoles. The compartment specific staining by led also differs from the generalized staining in all compartments given by uranyl. Thus the contents of luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC from the generalized staining in all compartments given by uranyl. Thus the contents of luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC from the generalized staining in all compartments given by uranyl. Thus the contents of luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC cisternae are devoid of uranyl stainable material. However, lead staining and acid phosphatase activity in the GC continue. We presume that they mark the environment within these cisternae rather than the proteins passing through them. This environment is itself not static. Several observations suggest that the function of cisternae that is detectable by lead staining is temporally discontinuous and related to a stage of maturation or development. Only early stage ims stain: the staining ceases by the beginning of autophagy after hydrolytic enzymes are presumed to have been added. Condensing vacuoles cease to stain as the central core crystallizes out. Stain may be absent from one or two GC saccules at any position in the stack as though the phase of lead staining (or lack or it) can move progressively through the system. We conclude that in studies characterizing components of the vacuolar system it is necessary to separate those that mark transient occupants of a compartment from those that mark the compartment itself. Both may vary temporally independently from one another.

Acid Phosphatase↗

PubMed2004

Differentiation-associated staining with anti-pimonidazole antibodies in head and neck tumors.

BACKGROUND AND PURPOSE: Hypoxia is a strong negative prognostic factor for all three major treatment modalities for cancer. The bioreductive drug pimonidazole is currently under clinical investigation as a hypoxia marker. In human head and neck tumors, in addition to staining patterns typical of chronic hypoxia, staining was seen specifically around areas of keratinization, raising the question of whether this is hypoxia-related. This could influence quantitative hypoxia estimates using this marker. We investigated here whether the differentiation-related staining was caused by locally high reductive enzyme levels. PATIENTS AND METHODS: The nitrotetrazolium compound NBT was used, which is reduced by nitroreductases to yield a blue color. The assay was validated on three genetically related MDA231 human mammary carcinoma cell lines: wildtype, overexpressing DT-diaphorase (DT1), and overexpressing cytochrome p450 reductase (R4). Increased NBT staining under normoxia was indeed seen for both R4 and DT1 lines. Pimonidazole staining under normoxia was only seen in the R4 line. RESULTS: Frozen tumor sections from 20 patients with head and neck cancer injected with pimonidazole were incubated with NBT. Parallel sections were stained for pimonidazole. Staining patterns were then compared on matched images, and areas of keratinization scored for the presence or absence of pimonidazole and NBT. Pimonidazole staining was seen in 56% of keratinized areas, and of these, 78% showed increased NBT staining, indicating that high reductase levels are not a necessary requirement for differentiation-associated pimonidazole staining. In a second series, frozen sections of tumors from 15 patients not receiving pimonidazole were incubated with NBT and compared with staining after incubation with pimonidazole under both oxic and hypoxic conditions. Pimonidazole staining of some keratinizing areas under oxic conditions was seen. Of these areas, only a proportion (70%) showed increased NBT staining, confirming the lack of correspondence between keratin-associated pimonidazole staining and reductase levels. CONCLUSION: Hypoxia-independent pimonidazole staining can occur in more differentiated head and neck tumors, necessitating caution in hypoxia quantification. These data argue against a causative role for locally high reductase levels in differentiation-associated staining. DT-diaphorase appears to play no role in pimonidazole reduction.

Antibodies, Anti-Idiotypic↗

PubMed2002

Corneal staining in hydrogel lens wearers.

PURPOSE: The purpose of this study was to determine the factors that contribute to corneal staining in hydrogel lens wearers by examining the following variables: type of lens worn, wearing time and lens replacement schedule, lens care system, and topical and systemic medication use. METHODS: Five hundred full-time, successful hydrogel contact lens wearers were evaluated for corneal fluorescein staining at 20 clinical sites. Corneal staining was evaluated in five regions and was graded on a scale of 0 (no staining) to 4 (severe staining) in 0.5 steps for severity. RESULTS: Corneal fluorescein staining was present in at least one eye in 55.7% of the hydrogel lens wearers examined in this study, and 8.0% had moderate-to-severe staining (defined as a cumulative staining score of > or = 3 with at least one quadrant score > or = 2). Staining was most severe for the inferior portion of the cornea (mean +/- SD; OD: 0.34 +/- 0.57, OS: 0.30 +/- 0.54). All other areas of the cornea showed lesser amounts of staining, all of which were approximately equal (mean range, 0.13 to 0.16). Staining was observed in two or more quadrants in 24% of right eyes and 22.4% of left eyes. When questioned about care system compliance, 81.1% of subjects reported compliance. Patients who were noncompliant with their lens care system (chi 2 p = 0.0037), used rewetting drops (chi 2 p = 0.0005), or wore conventional lenses without a planned replacement schedule (chi 2 p = 0.0008) were more likely to have some degree of corneal staining. Noncompliance with care system (chi 2 p = 0.0147), replacement schedule (chi 2 p = 0.0039), and lens power (chi 2 p = 0.0224) were associated with moderate-to-severe staining. The following factors were not significantly associated with corneal staining: patient age, gender, medication use including oral contraceptives, type of wear (extended, flexible, or daily), average wearing time, wearing time at the examination, lens type (spherical or toric), water content of lenses (high or low water content), type of care system, use of enzymes, and pinching the contact lens directly off the cornea. CONCLUSIONS: Staining occurs to some extent in many hydrogel contact lens patients, and is influenced by many factors. Moderate-to-severe staining, which may be more clinically significant, is associated with noncompliance with care system, a conventional replacement schedule, and lens power.

Adult↗

PubMed1997

A new staining method for identification of Helicobacter pylori and simultaneous visualization of gastric morphologic features.

Helicobacter pylori (HP) is prevalent in the general population and is associated with chronic active gastritis, peptic ulcers, gastric carcinoma, and lymphoma. Different methods to diagnose HP colonization include the urea breath test, serologic analysis, and gastric biopsy. Many different staining methods, including silver and Giemsa-based stains, have been used to demonstrate these organisms in gastric biopsy specimens. The tissue morphologic features, however, are often obscured, thus mandating evaluation of both hematoxylin and eosin (H&E) and special stains. This study evaluates the ability of a new staining method that allows simultaneous identification of HP and visualization of tissue morphologic features. We examined formalin-fixed, paraffin-embedded archival tissues from 184 consecutive gastric biopsy specimens using our new staining method, and we compared our results with those previously obtained from H&E and Warthin-Starry (WS) stains. Our new stain uses periodic acid-Schiff, Coleman's Feulgen solution, Mayer's hematoxylin, and methylene blue. Our method is technically simpler than the WS stain and can be completed in approximately 9 minutes. We found HP organisms in 76 (41%) of 184 biopsy specimens by H&E and/or WS staining. Our new staining method identified HP in 83 specimens (45%). The organisms stained bright blue and were easily visualized (compared with the H&E-stained specimens) because of the contrasting magenta mucin. Tissue morphologic features consisted of well-defined cells with dark blue nuclei and pale cytoplasm. Intestinal metaplasia was highlighted magenta Our new staining method revealed chronic active gastritis in 71 cases (39%), intestinal metaplasia in 18 cases (10%), and lymphoid aggregates in 35 cases (19%). These findings were similar to the results obtained with H&E-stained sections. We conclude that our new staining method is inexpensive, quick, and easy to perform and interpret. It has a sensitivity comparable to that of conventional staining methods and simultaneously demonstrates both tissue morphologic features and the presence of HP.

Biopsy↗

PubMedDate not supplied

Staining of non-carious human coronal dentin by caries dyes.

This study tested the hypothesis that commercially available caries dyes stain non-carious human coronal dentin in freshly extracted teeth. Multiple sections were cut from 10 non-carious and two control carious teeth using a water-cooled saw. Each section was stained with one of five caries dyes. The location of staining, if any, was noted and the staining intensity was scored on a four-point scale. One of the sections from each tooth was subsequently decalcified and processed for observation under a light microscope using four histologic staining techniques to evaluate morphology, collagen distribution and bacterial content. The association between the stain intensity scores on the undecalcified sections and the five dyes was evaluated using the Kruskal-Wallis One-Way ANOVA by Ranks test. Outer carious dentin in the control specimens stained intensely with each of the five dyes. In the undecalcified, non-carious sections, all had at least one area of staining. However, this staining could be differentiated from the intensity of dye staining in the carious controls, except in two instances. The association between stain intensity scores and the five dyes was not statistically significant. In the histologic sections, numerous bacteria were seen within the dentinal tubules of carious lesions of the two control specimens; however, no bacteria were found in any of the sections from non-carious specimens. Histologically, no differences were observed in the morphology or staining pattern within mantle or circumpulpal dentin in areas stained with caries dye, and in only one unique instance within the main body of the dentin. These results suggest that the five dyes evaluated in this study can stain non-carious dentin, however, this stain can be differentiated from the staining of outer carious dentin in vitro.

Analysis of Variance↗

PubMed1991

Combined Fontana-Masson-mucin staining of Cryptococcus neoformans.

Cryptococci react positively with various histochemical stains, including the Fontana-Masson (FM), which stains the cell wall, and mucin stains, such as alcian blue and mucicarmine, which stain the capsule. Combinations of the FM stain with both the alcian blue and mucicarmine stains were performed on paraffin-embedded tissue specimens that were obtained from 15 patients who had culture-proved cryptococcosis. Combined FM-mucicarmine and FM-alcian blue stains were compared with other individual fungal stains. The FM stain, followed by either the mucicarmine or alcian blue stain, distinctively demonstrated both the cell wall and capsule of most organisms. More organisms were recognized in the combined stains than with either stain done individually. No interference between the stains was noted. Combining the FM stain with either of these two mucin stains appears to be helpful for identifying cryptococci.

Alcian Blue↗

PubMed2001

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space.

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.

Coloring Agents↗

PubMed2005

Staining properties of deepithelialized human amniotic membrane.

PURPOSE: The surgical use of human amniotic membrane (HAM) can be challenging because of its transparency, thin structure, and adhesive quality. Staining of HAM has been proposed to facilitate intraoperative visualization. We evaluated the in vitro staining properties of deepithelialized HAM using 5 vital dyes. METHODS: Deepithelialized HAM was stained with indocyanine green (1.0%, 0.5%, 0.1%), fluorescein sodium (1.0%, 0.25%, 0.1%), lissamine green (0.5%, 0.1%), rose bengal (1.0%, 0.1%), and trypan blue (0.5%, 0.1%). The staining of each dyed sample was evaluated using a x10 operating microscope by a single observer. Each membrane was then rinsed, resuspended (3 mL of balanced salt solution), and then reevaluated for staining at 30-minute intervals for a total of 4 hours. All remaining stained samples were then placed in 5 mL balanced salt solution and then reevaluated for staining at 24 hours. RESULTS: All concentrations of the 5 dyes stained the membrane initially. After 120 minutes, fluorescein sodium 0.1% and lissamine green 0.5% and 0.1% no longer stained the membrane. Fluorescein sodium 0.5% no longer stained at 210 minutes, and fluorescein sodium 1.0% no longer stained at 24 hours. All concentrations of indocyanine green, rose bengal, and trypan blue stained positively at 24 hours. CONCLUSIONS: All 5 dyes stain deepithelialized HAM initially. Tested concentrations of fluorescein sodium and lissamine green may be superior to other tested dyes for intraoperative use because these dyes stain the membrane and then fade. Tested concentrations of rose bengal, trypan blue, and ICG may not be ideal for clinical use because they demonstrate persistent staining at 24 hours.

Amnion↗

PubMed1995

Comparison of three staining methods for detecting microsporidia in fluids.

Calcofluor white 2MR, modified trichrome blue, and indirect immunofluorescent antibody (IFA) staining methods were evaluated and compared for detecting microsporidia in stool. Serial 10-fold dilutions of Encephalitozoon (Septata) intestinalis were prepared in three formalinized stool specimens or in Tris-buffered saline. Ten-microliter aliquots were smeared onto glass slides, fixed with methanol, stained, and read by at least three individuals. The results indicated that the calcofluor stain was the most sensitive method, required approximately 15 min to perform, but did generate some false-positive results due to similarly staining small yeast cells. The modified trichrome blue stain was nearly as sensitive as the calcofluor stain and allowed for easier distinction between microsporidia and yeast cells. This stain, however, required approximately 60 min to perform. The IFA stain with polyclonal murine antiserum against E. intestinalis was the least sensitive of the methods and required approximately 130 min to perform. The lower limit of detection with the calcofluor and modified trichrome stains was a concentration of about 500 organisms in 10 microliters of stool to detect one microsporidian after viewing 50 fields at a final magnification of x1,000. Reliability was also addressed by use of 74 stool, urine, and intestinal fluid specimens, 50 of which were confirmed for the presence of microsporidia by transmission electron microscopy (TEM). All TEM-positive specimens were detected by calcofluor and modified trichrome blue staining. Ten specimens were not detected by the IFA stain. An additional seven TEM-negative specimens were read positive for microsporidia with the calcofluor stain, and of these, five also were read positive with the modified trichrome blue stain. The resulting diagnostic paradigm was to screen specimens with the calcofluor stain and to confirm the results with the modified trichrome stain. IFA, which was less sensitive, may become useful for microsporidian species identification as specific antibodies become available.

AIDS-Related Opportunistic Infections↗

PubMed1988

Fluorescent vital stains for complementary labelling of protoplasts from Trichoderma spp.

In this study several fluorescent vital stains were evaluated for their ability to provide complementary vital staining of protoplasts of Trichoderma spp. for selection of heterokaryons following protoplast fusion. Tetramethyl rhodamine isothiocyanate and fluorescein isothiocyanate were rejected because they stained only a small proportion of protoplasts. Fluorescein diacetate stained all protoplasts, but the chromophore leaked rapidly from stained cells. A mixture of FluoroBora T and acriflavine stained all cells, but intensity was low and fading upon illumination was rapid. Nile red stained lipid bodies in all cells, but the stain was lost upon protoplast fusion in polyethylene glycol. Rhodamine 6G, on the other hand, stained all cells, fluoresced green, and was stable through fusion and upon illumination. Hydroethidine also stained all protoplasts, and staining was relatively stable through fusion and upon illumination. Hydroethidine fluoresced red and stained nuclei more prominently than the cytoplasm. Rhodamine 6G and hydroethidine were tested on a number of strains to determine whether they were toxic to protoplasts. No toxicity to any strain was noted with rhodamine 6G. Hydroethidine, however, was toxic at the higher concentrations tested, especially when stained protoplasts were exposed to light. When protoplasts were stained with the minimum concentration giving ready visualization and were incubated in darkness, hydroethidine also was nontoxic. Hydroethidine and rhodamine 6G are useful complementary vital stains of Trichoderma protoplasts for visualization of frequency and type (dicell, multicell) of fusion.

Fluorescent Dyes↗

PubMed1988

Aldehyde pararosanilin (true aldehyde fuchsin) stains purified insulin, oxidized or not, following adequate fixation with either formalin or Bouin's fluid.

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from "basic fuchsin" whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC. Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content. We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.

Acetates↗

PubMed2004

The use of a differential fluorescent staining method to detect bacteriuria.

BACKGROUND: This report describes a differential staining method which distinguishes gram-positive from gram-negative bacteria in fluorescence. Gram-positive bacteria appear yellow and gram-negative bacteria appear green. MATERIAL AND METHODS: The method is based on two fluorochromes, one acting in the wavelength of red, i.e. the acridine orange, and another acting in the wavelength of green, i.e. the fluorescein, which together form a red/ green system. In this report we compared the accuracy of the differential fluorescent staining method and the Gram stain in screening for bacteriuria, as detected by conventional cultures. RESULTS: A total of 1487 urine samples were tested. 289 cultures were positive. 237 specimens grew a single organism at 10(5) and 10(4) CFU/ml. 224 smears were detected by the differential fluorescent staining method and 162 were detected by Gram stain. 1198 samples failed to grow organisms at 10(5) and 10(4) CFU/ml. 107 smears were falsely positive by the fluorescent staining procedure and 289 were falsely positive by the Gram stain. On the basis of the culture results, the sensitivity of the differential fluorescent staining method was 94.5% and that of the Gram stain 68.3%. The specificity of the fluorescent staining procedure was 91.6% and that of the Gram stain 75.8%. The positive predictive value and the negative predictive value of the fluorescent staining method were 67.6% and 98.8%, respectively. Those of the Gram stain were 35.9% and 92.3%, respectively. CONCLUSIONS: A wide range of microbiological and chemical techniques are available to identify bacteria in urine. This fluorescent staining method represents a simple, rapid, reliable method with low-running costs. The main advantage of this technique is that it enables the microbiologist to exclude the presence of bacteria in the urine within a short time after specimen receipt and to eliminate a large number of specimens for culture with significant cost saving. Another advantage of the method is that it allows to distinguish gram-positive from gram-negative bacteria in positive slides on the same day the sample is obtained. The stained smears were easily interpreted, even when the bacterial counts in the specimen were low.

Acridine Orange↗