A comparison of the highly selective fluorescence staining of fungi in tissue sections with Uvitex 2B and Calcofluor White M2R

Summary The selective fluorescence staining of two fungi,Candida albicans andBlastomyces dermatitides, with Uvitex 2B and Calcofluor White M2R was studied in deparaffinized and frozen sections of mouse kidney and lung. Both fluorochromes emitted maximally at about 430nm, independent of the mounting media (Kaiser's gelatin or Entellan). In addition to fungi, both fluorochromes also stained elastic fibres. The fluorescence intensity remained unchanged after storage of sections for more than 6 months in conventional slide boxes. the two fluorochromes showed the following differences: Calcofluor faded 1.25 times faster than Uvitex when illuminated with ultraviolet light. Calcofluor showed a greater affinity for tissues in general, and red cells and renal tubular casts in particular. Counterstaining of deparaffinized sections with Hemalum and Eosin reduced the fungi fluorescence and suppressed the general background fluorescence. However, it led to an intensification of Eosin staining and the fluorescence of red cells in Calcofluorstained sections but not in Uvitex-stained ones. Similarly, the background fluorescence in frozen sections was reduced by Evans Blue, although elastic fibres still fluoresced after staining with Calcofluor. The degree of staining selectivity, and thus the contrast produced within a histological specimen, was greater with Uvitex 2B than with Calcofluor White M2R.     

Eosin-related fluorescence of acidophil pituitary cells

The examination of haematoxylin and eosin stained sections of normal and neoplastic pituitary glands under ultraviolet light illumination discloses fluorescence of acidophil cells. The distinction between prolactin and growth hormone-producing cells is not possible. Such fluorescence depends on previous eosin staining.     

Phosphotungstic Acid-Eosin Combined with Hematoxylin as A Stain for Negri Bodies in Paraffin Sections

Experimentation with the Papanicolaou stain in this laboratory led to the discovery that the eosin, combined with phosphotungstic acid, was responsible for differential staining of Negri bodies. Eosin prepared as in EA 36 was used, but without the light green and Bismarck brown. Paraffin sections of hippocampus from brains of animal affected with rabies were fixed in 10% formol or in a mixture of 2 volumes of saturated aqueous HgCl₂ and 1 volume of absolute alcohol. They were stained first with hematoxylin and then with eosin. This procedure gave better results than staining with other types of eosin or by the original EA 36 mixture. The Negri bodies were well stained and their structure easily visible. The best results were obtained from material fixed with the HgCl₂ solution.     

The Combined Perls-Hematoxylin-Eosin Stain

To provide a routine check for the presence of ferric iron in sections, Perls' method was combined with hematoxylin and eosin as follows. Deparaffinized sections of formalin-fixed tissues are stained in Perls' reagent (1:1 2%, w/v, of potassium ferrocyanide in distilled water and 2%, v/v, concentrated HCl in distilled water) for 20 min. After brief rinsing in distilled water stain sections in Mayer's hemalum, wash in tap water for 5 min, counterstain in 0.5% (w/v) eosin B in 50% ethyl alcohol for 15 sec. Rinse in tap water, dehydrate and mount as usual.     

Selective fluorescence of eosinophilic structures in grasshopper and mammalian testis stained with haematoxylin-eosin

After staining with Mayer's haematoxylin and eosin Y, paraffin sections of grasshopper and mouse testis were analysed by both transmitted light and fluorescence microscopy. Under violet-blue (436 nm) light excitation, a bright green emission was observed in all eosinophilic structures. Meiotic spindles (fibres and poles), mitochondrial aggregates, centriolar adjuncts in grasshopper spermatids, the basal lamina, flagellar bundles and remaining cytoplasmic droplets in the lumen of seminiferous tubules showed the most striking fluorescence induced by eosin Y. No emission was found in these structures after haemalum staining. Fluorescent microtubular components also revealed a positive immunoperoxidase reaction for -tubulin. All fixation and embedding procedures (Bouin, Zenker, formaldehyde alone or followed by dichromate or glutaraldehyde, freeze-substitution) were suitable for observation by fluorescence microscopy. Acetylation, deamination, and prolonged washing of stained sections with water, salt solution or ethanol strongly reduced eosin Y fluorescence, while it slightly increased after methylation. These results show that routine haematoxylin-eosin stained tissue sections can be routinely analysed by fluorescence microscopy. The emission of eosin Y allows easy and precise recognition of eosinophilic structures, which are poorly visible under bright field illumination.     

Staining of fungal cell walls with fluorescent brighteners: Flow-cytometric analysis

Spore walls ofBackusella lamprospora (Mucorales) were stained with ten fluorescent brighteners (FB) and the intensity of their fluorescence was determined. The fluorescence was most intense with Uvitex 2B (100%), other brighteners yielding lower fluorescence intensities: Blankophor BA 267% and BA 200% about 75%, Rylux BSU about 50%, other Rylux agents 10–30%. The agents most suitable for microscopic diagnostics of human and animal mycoses are Uvitex 2B, Blankophor BA 267% and BA 200%, Rylux BSU, and also Rylux BS and PRS. The regulation of excessive fluorescence of fungal cells during microscopic observation is discussed. For the purposes of microscopic diagnosis of human and animal mycosis Uvitex 2B, Blankophor BA 267% and BA 200%, Rylux BSU and, possibly, Rylux BS and PRS are recommended.     

Fluorescent staining of elastic tissue with Rhodamine B and related xanthene dyes

Summary Frozen sections, cut at 8 from various fresh tissues, were dried for 5 minutes over sulfuric acid, and then stained for 4 minutes in a 0.1% aqueous solution of Rhodamine B (Chroma) at pH 8.O. After soaking twice in butanol for 2 minutes each, the sections were kept on a warmer at 60° C for 2 minutes and then mounted in oil of cedar. Under ultraviolet microscopy, elastic fibers selectively stained yellow-orange against a pale blue background. No autofluorescence of elastic fibers was observed in guinea pig, rat, hamster or rabbit tissue in contrast to the autofluorescence of elastic fibers typically seen in human tissue.Similar fluorescent staining of the elastic tissue could be achieved by using a number of related xanthene dyes including pyronine, eosin B, acridine orange, pholoxin, phloxin B. More distantly related auranine O and thiazol yellow G were likewise used with success as fluorochromes for elastic fibers.Supported by a John A. Hartford Foundation Grant.     

Taenia taeniaeformis: early inflammatory response around developing metacestodes in the liver of resistant and susceptible mice II. Histochemistry and cytochemistry

Female BALB/cJ (resistant), C3H/HeJ (intermediate resistant), and C3H/HeDub (susceptible) inbred mice, 4-5 wk old, were infected with Taenia taeniaeformis. Liver sections were stained for the enzymes acid phosphatase, beta-glucuronidase, and peroxidase. Eosinophils present around the parasite were identified by the ethanolic Congo red method. Possible gross changes in lipid metabolism in the hepatocytes surrounding the parasite were investigated with the Sudan black B method. The results of observations made by light microscopy were: (1) beta-glucuronidase activity above background levels was observed only in the hepatocytes around the parasite in BALB/cJ mice at 4, 5, and 6 days postinfection (PI); no reaction was observed in the other 2 strains of mice studied; (2) acid phosphatase activity was very strong at 2, 3, and 4 in the 3 strains of mice while this reactivity was weak at 5 and 6 days PI; (3) the cytoplasm of the hepatocytes around the metacestode stained more heavily with Sudan black B than other hepatocytes; and (4) the presence of eosinophils appearing at 3 days PI around the parasite in all 3 strains of mice was demonstrated by staining with Sudan black B, the substrate of peroxidase, and Congo red. Infected C3H/HeJ and BALB/cJ mice had higher numbers of liver eosinophils than infected C3H/HeDub mice throughout the observation time. The present results suggest 2 conclusions: (1) a parasite-liver interaction occurs as is evident by hepatocyte changes in beta-glucuronidase activity and Sudan black B staining, and (2) resistance to the early stages of T. taeniaeformis is associated with the appearance of eosinophils.     

Pathological effects of Echinostoma caproni (Trematoda) in the domestic chick

Domestic chicks experimentally infected with Echinostoma caproni for 2 weeks showed a dilated ileum, unkempt feathers, watery diarrhoea, and weight loss. The ileum from infected and control chicks was fixed in 10% neutral buffered formalin, and prepared as 10 microns paraffin sections stained in haematoxylin and eosin, Papanicolau, periodic acid-Schiff, picro-ponceau, and alcian blue. The infected ileum showed atrophic villi, hypertrophied circular musculature with collagen-like fibres and haemorrhagic zones. The brush borders of epithelial cells and goblet cells were absent in the mucosa of the infected ileum. Worms in contact with host mucosa showed tissue plugs in the suckers. The cephalic spines of worms abraded the host mucosa.     

Direct Staining of Reticular Fibers with Gold Chloride

Reticular fibers are selectively stained in paraffin sections of formalin-fixed or Bouin's-fixed tissue as follows: 1% aqueous solution of gold chloride for 20 min, followed by a 10 min immersion in an aqueous solution containing 5% Na₂CO₃ and 0.5% KOH. The sections then are placed in a 5% aqueous solution of KI for 2 min. Counterstaining with a 0.25% aqueous solution of methylene blue chloride is optional. The reticular fibers stain dark pink; the collagen bundles are a light pink to straw color without the counterstain, or a light blue color when the methylene blue is used.     

The application of immunoperoxidase staining for the detection of causative fungi in tissue specimens of mycosis I

This study was performed in order to identify the fungi of four species (Aspergillus fumigatus, Fusarium anthophilum, Candida albicans, Cryptococcus neoformans) in formalin-fixed, paraffin-embedded tissue sections by the indirect method of immunoperoxidase staining. Mature albino rabbits were immunized by formalin-killed organisms. The antibodies were prepared by precipitation at a 50% saturation of ammonium sulfate and were checked for cross-reactivities by Ouchterlony's double immunodiffusion and precipitin test. The immunoperoxidase staining was applied to the paraffin-embedded tissue sections of infected mice, human autopsy and biopsy specimens. Although each fungus was stained clearly the cell wall, cross-reactivities appeared among them, however it was possible to identify four fungi by absorption and dilution of the antisera.     

Camwood (Baphia nitida) Extract as a Histological Stain for Interglobular Dentine, Striated Muscle, and Counterstaining

The shavings of the dried heartwood of the tree Baphia nitida are ground to a fine powder, and 6 gm of the powder are extracted in 100 ml absolute ethanol at 27-30 for 6-24 hr. The extract is filtered with Whatman No. 1 paper and stored in a screw-capped bottle. For staining the interglobular dentine of nondecalcified sections of formlin-fixed teeth, sawed cross sections 20-30 μ thick were dehydrated in ethanol and stained in the undiluted extract for 6-12 hr at room temperature. The interglobular dentine was stained a bright golden brown on a pale brown background. For staining striated muscle, the extract was diluted 1:1 with distilled water and filtered. After mordanting formalin-fixed paraffin sections with 0.25% KMnO₄ for 5 min, and bleaching with 5% oxalic acid for 10 min, they were washed in water and stained for 2-24 hr at room temperature. The striations were stained light to deep golden brown. For use as a counterstain, a 1:6 dilution of the original extract was required. When applied after haematoxylin for 15-30 min, it stained tissue components in varying shades of golden brown with distribution comparable to that produced by 1% eosin.     

Taenia taeniaeformis: early inflammatory response around developing metacestodes in the liver of resistant and susceptible mice I. Identification of leukocyte response with monoclonal antibodies

Female BALB/cJ (resistant), C3H/HeJ (intermediate resistant), and C3H/HeDub (susceptible) inbred mice, 4-5 wk old, were infected with Taenia taeniaeformis. Liver sections were stained by an immunoperoxidase technique (avidin-biotin complex, ABC) for the differentiation antigens Lyt-1, Lyt-2, Mac-1, Mac-2, Mac-3, and B220. Binding of ABC to the cytoplasm of hepatocytes around the developing parasite was observed at 4 days postinfection (PI) in all 3 strains of mice, persisting in BALB/cJ and C3H/HeJ liver sections at 5 and 6 days PI, suggesting the presence of high concentrations of biotin, a fatty acid synthesis mediator. Two cell populations were labeled with B220 monoclonal antibodies: lymphocytes and polymorphonuclear (PMN) cells. At 4 days PI the number of labeled PMN cells peaked in infected C3H/HeJ and BALB/cJ mice; however a low number of PMN cells were labeled in infected C3H/HeDub mice. Few lymphocytes bound the B220 antibody in either BALB/cJ, C3H/HeJ, or C3H/HeDub infected mice. The number of Mac-1+ cells detected in infected C3H/HeJ and BALB/cJ liver sections were similar whereas fewer Mac-1+ cells were present in infected C3H/HeDub mice. Mac-2+ cells appeared in high numbers around the growing parasite at 5 and 6 days PI in the liver of C3H/HeDub mice, but not in the liver of BALB/cJ mice. Mac-3+ cells followed a similar pattern to that of the cell population defined by Mac-2. Few Lyt-1+ and Lyt-2+ cells were detected around the parasite site in the 3 strains of mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

A STAINING TECHNIQUE FOR AUTORADIOGRAPHS OF NONDIFFUSIBLE ISOTOPES

Nondeparaffinized radioactive tissue sections are stained with hematoxylin and eosin by being floated on aqueous solutions for 1 hr each. The sections are then thoroughly washed, dried and exposed to autoradiographic plates or emulsions for predetermined periods of time. When desirable, both stained and unstained adjacent tissue sections can be mounted on a single slide of autoradiographic plate for exposure. Kodak DK-19 and 30% Na₂S₂O₃.5H₂O solutions are used for subsequent developing and fixing. The finished autoradiographs show excellent resolution and cytologic detail, since the gelatin remains unstained while the tissue retains its stain. Stains other than hematoxylin and eosin can be applied to this technique, provided they withstand the developmental and fixation processes.     

Staining of Pathogenic Fungi with Gomori's Aldehyde-Fuchsin in Both Old and New Sections

Gomori's stain for elastic tissue can be applied directly to paraffin sections as a routine method for fungus capsules and membranes. Either freshly prepared, or old sections previously stained with hematoxylin-eosin, can be used. After applying the aldehydefuchsin stain, nuclei may be stained (or restained) with hematoxylin, and a general background stain obtained with either eosin or light green.     

Picrosirius Red Staining of Dental Structures

Histologic sections of dog mandibles and teeth were stained with picrosirius red and Mayer's hematoxylin. Collagenous structures of the mandible stained brilliant red. Dentinal tubules, Sharpey's fibers and other structures not easily seen in sections stained with hematoxylin and eosin alone were seen clearly after this procedure. Under polarized light collagen fibers could be specifically identified and their orientation determined. Picrosirius red-hematoxylin is recommended for examination of normal or pathologic dental specimens.     

Picrosirius red staining of dental structures

Histologic sections of dog mandibles and teeth were stained with picrosirius red and Mayer's hematoxylin. Collagenous structures of the mandible stained brilliant red. Dentinal tubules, Sharpey's fibers and other structures not easily seen in sections stained with hematoxylin and eosin alone were seen clearly after this procedure. Under polarized light collagen fibers could be specifically identified and their orientation determined. Picrosirius red-hematoxylin is recommended for examination of normal or pathologic dental specimens.     

Infección múltiple fúngica en un paciente diabético

BackgroundMixed fungal infections although undervalued, are more common than mentioned in the scientific literature. These infections have a poor prognosis for the patient.ObjectivesWe present an unusual case of a 61-year-old diabetic male who had a rhino-orbito-sinusal zygomycosis in 2001. After surgical debridement of the infected parts, along with antifungal therapy with liposomal amphotericin B, the patient started improving. Several years later the patient was hospitalized due to a similar problem and was diagnosed of rhino-orbito-cerebral zygomycosis.MethodsIn both episodes, a histopathological examination and cultures were performed on the sinus lesions. Tissue sections were stained with haematoxylin and eosin, Giemsa, periodic acid-Schiff (PAS) and Grocott's methenamine silver, and cultures specific for fungi were performed.ResultsThe histopathology studies revealed the presence of bacteria, actinomyces and a mixed infection by at least four different fungi, all of them well differentiated by their morphology. Despite the rapid diagnosis the patient died due to spreading to the central nervous system.ConclusionsMixed infections by fungi are rare, but due to the high incidence of immunodeficiencies they could occur more often than reported. We would like to alert on the possibility of acquired mixed infection by fungi which have shown to be high aggressive and have a worse prognostic in patients with underlying diseases.     

Keratin in the Egg Shells of Amphistomes; Histochemical Differentiation from Quinonetanned Protein in Other Trematoda

Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H₂SO₄ for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H₂SO₄ and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.     

Procion Brilliant Orange, A Counters Iain for Decalcified Sections Vitally Labeled with Procion Brilliant Red H-8 Bs

Procion brilliant red H-8 BS is a fluorescent dye that stains the organic matrix of hone supravitally. A procedure is described for counterstaining sections of such hone for visible light examination without interfering with the demonstration of sites of hound dye under UV illumination. Sections are brought to water, stained in Delafield's alum hematoxylin for 10 minutes, washed in tap water for 10 minutes, counterstained in 1% Procion brilliant orange M-GS for 15 minutes and washed in distilled water for 10 minutes. After dehydration the sections were mounted in Eukitt.