A Basic Fuchsin and Alkalinized Methylene Blue Rapid Stain for Epoxyembedded Tissue

Sections 1 μ thick of epoxy-embedded, OsO₄-fixed tissues were stained with 4% aqueous basic fuchsin at 70 C for 1 min, rinsed well and destained, also at 70 C, for 1 min. A 2% aqueous methylene blue solution, alkalinized to pH 12.5 by mixing 1 N NaOH with the dye on the slide in the proportion of about 2:1, was then allowed to act for 2 min at 23-27 C. The stain was rinsed off the slide, and the preparation air dried before applying a mounting medium and cover glass. The mounting medium consisted of immersion oil sealed with epoxy household cement. Stains had not faded after 1 yr. The method is simple, rapid (total time 4-5 min), and provides sharp contrast between cellular and connective tissue components.     

Sequential Use of Wright's and Ziehl-Neelsen's Stains for Demonstrating Phagocytosis of Bacterial Spores

Nongerminating spores, germinating spores, and vegetative cells of Clostridium botulinum type A were observed during phagocytosis in the peritoneal fluid of white mice. Since phagocytes are easily ruptured by heat, the method described avoids heating, as this has been employed in conventional spore staining methods. A thin smear of the fluid is air dried on the slide for 2 hr, and stained by Wright's method: stain, 2 min; dilution water, 2 min; and rinsed; then in 0.005% methylene blue for 30 sec, and rinsed. This is followed by Ziehl-Neelsen's stain for 3-4 min and destained with 1: acetone-95% ethanol for 10 sec. The slide is rinsed, and Wright's staining repeated: stain 1 min, dilution 2-3 min; and thereafter washed about 5 ml of Wright's buffer. Blotting and air drying completes the staining. Non-germinating spores stain light red with a red spore wall, germinating spores are deep red throughout, vegetative cells are blue, and leucocytes show a dark purple nucleus and light blue cytoplasm.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

Basic Two-Dye Stains for Epoxy-Embedded 0.3-1 μ Sections

Dyes used in the 3 methods recommended are: I, thionin and acridine orange (T-AO); II, Janus green and Darrow red (JG-DR); III, methyl green and methyl violet (MG-MV). The first 2 methods were two-solution stains, applied in sequence; the third, required only one solution since methyl violet is present in commercial methyl green. Staining solution and timing was as follows: Method I. 0.1% thionin in a 45% ethanolic solution of 0.01 N NaOH, 5 min at 70 C; rinsing in water and followed by 1 min in a 1% aqueous solution of acridine orange made up in 0.02 N NaOH, also at 70 C, then washed, and dried on slides. Method II. 0.5% Janus green in aqueous 0.05 N NaOH, 5 min at 70 C; rinsing in water then into 0.5% Darrow red in 0.05 N NaOH (aq.), 2 min at 70 C., washing, and drying on slides. Method III. 1% methyl green (commercial, unpurified) in 1% aqueous borax for 15-20 min at 20-25 C, washing and attaching to slides. All staining was performed by floating the sections on the staining solutions, all drying at 70 C, and mounting in a resinous medium. T-AO gave blue to violet cytoplasmic structures, darker nuclei which contrasted strongly with yellow connective tissue and the secretion of goblet cells. JG-DR resembled a hematoxylineosin stain, but by shortening the staining time in DR to 0.5-1 min, collagenous and elastic tissue retained more of the green dye. MG-MV gave dark green nuclei in light green cytoplasm, with collagenous and elastic tissues in blue to violet. As with most methods for staining ultrathin sections, thicknesses of less than 1 μ required longer staining times.     

Permanent Mounts Of Chromosomes After 8-Oxyquinoline and Squashing

Fresh young root tips or free-hand cross sections thereof were placed in 0.002 M 8-oxyquinoline (aq.) at 10-14^oC. for 3 hours. After rinsing in water 1-2 minutes, they were soaked in N HC1 at 55^oC. for 25 minutes, rinsed again and squashed under a cover glass on a dry slide. Slide and cover glass were separated by placing in 70% alcohol and allowed to remain therein at least 0.5 hour after separation. Both slide and cover glass were passed through 50% and 30% alcohol to water and stained by the Feulgen procedure (without further hydrolysis) or with crystal violet after mordanting in 1% chromic acid overnight and washing in running water 3-4 hours. Dehydration and mounting in balsam completed the process. The smear on the slide was covered with a clean cover glass and the cover glass, bearing stained material, mounted separately.     

Modified Whipf's Polychrome: A Connective Tissue Stain with Special Application for Demonstrating Leishmania

A polychrome stain procedure was developed to demonstrate amastigotes of the protozoan parasite Leishmania braziliensis as well as cytoplasmic and other tissue components in cutaneous lesions of infected animals. The procedure is as follows: stain nuclei for 10 minutes with an iron hematoxylin containing 0.5% hematoxylin and 0.75% ferric ammonium sulfate dissolved in 1:1 0.6 N H₂SO₄:95% ethanol; rinse 4 minutes in distilled water. Cytoplasmic staining is achieved by exposing tissues for 10 minutes to a solution containing 0.25% Biebrich scarlet, 0.45% orange G, 0.5% phosphomolybdic acid and 0.5% phosphotungstic acid in 1% aqueous acetic acid. These first two solutions are modified from Whipf's polychrome stain. Sections are differentiated for 10 seconds in 50% ethanol, rinsed in water, stained 3 minutes in 0.1% aniline blue WS in saturated aqueous picric acid, rinsed in water and differentiated for 1 minute in absolute ethanol containing 0.05% acetic acid. Mordanting overnight in 6% picric acid in 95% ethanol produced optimal results.

This procedure was applied to sectioned material from experimental animals with various protozoa. Trypanosoma cruzi, Besnoitia Jellisoni, Toxoplasma gondii and especially Leishmania braziliensis were well demonstrated. Combining cytoplasmic dyes and phosphomolybdic-phosphotungstic acids into one solution afforded differential staining of tissues by Biebrich scarlet and orange G; connective tissues were stained by this solution. Substantially improved definition of connective tissues resulted after counterstaining. This procedure differs from the Massou sequence in which connective tissues are first stained by cytoplasmic dyes along with other tissues and then destained prior to specific counter-staining. in comparing dyes structurally related to Biebrich scarlet, it was found that Crocein scarlet MOO, but not Poncenu S, was an acceptable substitute. Sirius supra blue GL and Sirius red FSBA were not useful as replacements for aniline blue WS in this procedure.     

Selective Staining of Protein and Starch in Wheat Flour and its Products

The main constituents of wheat flour and many wheat flour products are wheat protein (gluten) and starch granules. The specific staining of the protein present was effected by 10 min in 0.1% aqueous ponceau 2R (C.I. No. 16150) acidified with 3—4 drops of 1 N H₂SO₄ per 50 ml of staining solution, followed by rinsing in 2 changes of distilled water, dehydrating, clearing and mounting in a resinous medium in the normal way. Staining of starch was as follows: sections or flour smears were brought to water, treated for 10 min in a protein-blocking reagent (Taninol ADR—Imperial Chemical Industries—used in 1% aqueous solution) rinsed, then stained for 3 mins in 0.5% aqueous chlorazol violet R (C.I. No. 32445) or for 10 min in either 0.5% aqueous chlorazol violet N (C.I. No. 22570), or chlorazol black E (C.I. No. 30235). Staining was followed by thorough rinsing, normal dehydration and clearing and mounting in a medium of R.I. about 1.49 to enhance visibility of unstained starch grains. The methods are applicable to flour smears, cryostat and wax sections.     

A Phloxine-Azure-Hematoxylin Sequence for Differential Staining of Cells in Pancreatic Islets

Sections of 6 μ from tissues fixed in Susa or in Bouin's fluid (without acetic acid) and embedded in paraffin were attached to slides with Mayer's albumen, dried at 37 C for 12 hr, deparaffinized and hydrated. The sections fixed in Susa were transferred to a I₂-K₁ solution (1:2:300 ml of water); rinsed in water, decolorized in 5% Na₂S₂O₃; washed in running water, and rinsed in distilled water. Those fixed in Bouin's were transferred to 80% alcohol until decolorized, then rinsed in distilled water. All sections were stained in 1% aqueous phloxine, 10 min; rinsed in distilled water and transferred to 3% aqueous phosphotungstic acid, 1 min; rinsed in distilled water; stained 0.5 min in 0.05 azure II (Merck), washed in water; and finally, nuclear staining in Weigert's hematoxylin for 1 min was followed by a rinse in distilled water, rapid dehydration through alcohols, clearing in xylene and covering in balsam or a synthetic resin. In the completed stain, islet cells appear as follows: A cells, purple; B cells, weakly violet-blue; D cells, light blue with evident granules; exocrine cells, grayish blue with red granules.     

Stability of fungal α-amylase in sodium dodecylsulfate

Unfolding of a fungal -amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the -amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90°C for 10 min, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 min at various temperatures. The melting temperatures were 70, 55, and 49°C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the -amylase is weak. However, the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50°C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the -amylase is a slow process.     

Operational and storage stability of neutral β-mannanase from Bacillus licheniformis

The stability of neutral -mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l^–1 over 30 min at 25 °C. In an enzymatic membrane reactor operated at 50 °C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4 °C after one year.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

Stability of xylanases in the presence of methanol and its evaluation on the bleaching capacity

A commercial (Cartazyme) and non-commercial (Asperzyme) xylanases were studied. Cartazyme stability in a 0–70% (v/v) methanol at 50°C and 65°C was carried out. No deactivation was found for Cartazyme in the presence of 15% methanol at 50°C. Half-life activity decay (t1/2) of Cartazyme at 50°C in 30%, 50% and 70% methanol solutions were 4.0 h, 2.3 h and 1.2 h, respectively. At 65°C, which is the ozone-alkali-peroxide (ZEP) bleaching temperature, only significant results on Kappa number reduction and selectivity were only observed in 15% methanol (t1/2 30 min) at the Z stage. For the Asperzyme, a t1/2 of 36.5 min at 50°C was found. In the Z stage with Asperzyme in the presence of 25% of methanol, a 20% Kappa number reduction and an improvement of the ZEP sequence of the brightness of 3.1 points were obtained. These results were correlated with the xylanase stability.     

Short communication: Thermostability of α-amylase produced by Bacillus sp. E2—a thermophilic mutant

An -amylase from a hyper-producing strain of Bacillus (sp. E2) was stable at 70°C for 30 min but was quickly inactivated at higher temperatures. In the presence of 10mm Ca²⁺ and starch (20% w/v), however, the enzyme was stable at 90°C for 10 min and after 30 min at 100°C still retained 26% of its initial activity.     

Methodological aspects on immunohistochemistry in dermatology with special reference to neuronal markers

Summary In an attempt to optimize the immunohistochemical procedure for visualizing neuronal markers, such as neuropeptides, in the human skin, different alternatives in all steps of the process were compared. We have studied the influence of type of immunohistochemical method, the biopsy technique, including the size of the punch biopsy, anaesthesia, the choice of fixative and the time of fixation, the storage process, the sectioning parameters, incubation procedure, the type of fluorophore-conjugated antibody and its dilution, mounting and storage, and, finally, microscopical examination.The following procedure was found to give the best result: punch biopsies of 3 mm, taken under local anaesthesia using lidocaine injected into the dermis-subcutis at the place of biopsy; fixation by a buffered 10% formalin solution containing 14% of saturated picric acid for 2 h at 4°C; storage in 10% sucrose buffer for at least 24 h up to 1 month at 4°C or deep-frozen to -70°C for 2 months (with only a minor structural deterioration); cryostat sectioning of the biopsies with a section thickness of 14 m and with the cutting edge perpendicular to the skin surface; rhodamine (TRITC)-conjugated, instead of fluorescein-isothiocyanate (FITC)-conjugated, secondary antiserum, since it gives a lower background fluorescence; and for the incubation and mounting procedures, our standard laboratory routines were applied. The result is an optimal indirect immunofluorescence technique, to be applied in dermatology. We also found that biopsies taken under local anaesthesia with chlorethyl spray lost almost all immunofluorescence for several neuronal markers in the epidermis-upper dermis.     

Comparative studies of Feulgen hydrolysis for DNA

Summary We compared the Feulgen hydrolysis curves (37° C, 5 M HCl) of human blood lymphocytes fixed by the following four methods: 96% ethanol, 60 min at 20° C; ethanol-acetone, 11, 120 min at 4° C; ethanolglacial acetic acid mixture (31), containing 2% of formaldehyde (EAF), 90 min at 20° C; and ethanol-glacial acetic acid (31), 60 min followed by 5% chrome alum solution for 360 min at 20° C. The best results were obtained with EAF-fixation, with respect to the highest amount of DNA-Schiff complex at the peak point of the curve, the longest plateau region and the smallest scattering of DNA-Schiff amount values along the plateau. With other types of fixation the addition of polyethylene glycol (PEG) 6,000 to the hydrolysis solution resulted in modification of the shape of the hydrolysis curve so that it became nearly similar to the EAF-curve. The effect of PEG₆₀₀₀ on the EAF-curve was minimal. Slides fixed by ethanol were used for a comparison of polyethylene glycols with m.w. 1,500, 6,000 and 20,000. The longest plateau was obtained with PEG₆₀₀₀. The only effect of PEG₁₅₀₀ was a dramatic increase of DNA-Schiff amount at the peak point. PEG₂₀₀₀₀ had no significant effect on the shape of the hydrolysis curve. The results are discussed in terms of Kjellstrand's chain with a stable structure model of Feulgen hydrolysis.     

Differential staining of nerve cells and fibres for sections of paraffin-embedded material in mammalian central nervous system

A differential staining method is described of myelinated fibres and nerve cell bodies applicable to sections of mammalian, including human, central nervous system specimens embedded in paraffin wax. Experimental and human necropsy material fixed in acetic paraformaldehyde in phosphate buffer was used. Sections of 15–20 m in thickness were obtained, attached to slides, deparaffinized and hydrated. After hydration, sections are oxidized (30 s) in 2% potassium permanganate, bleached (1 min) in 5% oxalic acid and rinsed in distilled water. Staining is for 2–5 h in the following solution: 0.06% thionin, 1% formaldehyde, 10% acetic acid in distilled water. Sections are subsequently washed in distilled water, dehydrated through 96% and absolute ethanol, cleared in eucalyptol and mounted in Eukitt. Using the method described in the present paper, a differential coloration of myelin and neurons is obtained. Myelinated fibres appear red, whereas nerve cell bodies and glial nuclei are stained blue. This procedure provides a high contrast between myelin and cells suitable for observation and photography of sections. Simultaneous and differential coloration of both myelin and cells is easily and directly obtained with constant and homogeneous results.     

Isolation, purification and properties of a thermostable chitinase from an alkalophilic Bacillus sp. BG-11

An alkalophilic, chitinase-producing Bacillus sp. BG-11 was isolated which produced an extracellular chitinase and which was purified 16.5-fold, using standard purification techniques. The purified chitinase exhibited a broad pH and temperature optima of 7.5-9.0 and 45 deg C-55 deg C, respectively. The chitinase was stable between pH 6.0-9.0 and 50°C for more than 2 h. Half lives of enzyme at 60 deg C, 70 deg C and 80 deg C were 90 min, 30 min and 20 min respectively. Km value was 12 mg chitin per ml. Shelf life was 60 days at 4°C. Ca2+, Ni2+ and Triton-X-100 stimulated the activity up to 20% whereas Ag+, Hg2+, dithiothreitol, -mercaptoethanol, glutathione, iodoacetic acid and iodoacetamide inhibited the activity up to 50%.     

Improved in situ hybridization and G-banding by pretreatment with Denhardt''s solution and gelatin-chrome alum

Summary Various pretreatments of metaphase spreads were examined to obtain optimal DNA labelling patterns while maintaining chromosome integrity duringin situ hybridization procedures. Preparations of African green monkey (AGM) chromosomes fixed in methanol-acetic acid (CV-1 cell line) were treated by coating with Denhardt's solution, dilute gelatin-chrome alum, nonfat instant dry milk dissolved in saline—citrate solution (SSC) and/or acetylation prior to denaturation of chromosomal DNA in 70% formamide-2 x SSC for 2 min at 70° C. A³H-labelled, cloned DNA fragment of the highly, repetitive AGM component DNA was hybridized to the chromosomes by incubation at 45° C for 16 h. Treatment with gelatinchrome alum prior to denaturation greatly improved chromosome morphology and decreased background, but reduced pericentromeric labelling. Sequential treatment with 5 x Denhardt's solution followed by gelatin-chrome alum resulted in enhanced specificity of labelling and excellent chromosome morphology, as well as reduced levels of background. Acetylation had little effect after pretreatment with gelatin-chrome alum, but reduced background levels after pretreatment with Denhardt's solution. Chromosomes treated with Denhardt's solution plus gelatin-chrome alum can be routinely G-banded using trypsin afterin situ hybridization.     

Effect of high temperatures on seed germination of two woody Leguminosae

Cytisus scoparius and Genista florida regenerate after fire by stump-sprouting but also by seed. Seeds of these species were heated to a range of temperatures similar to those registered on the surface soil during natural fires (from 50 to 150 °C) and a range of exposure times (from 1 to 15 min). No germination was observed at high temperatures, 130 °C, when the exposure time was 5 min or more. However, moderate heat treatments (at 70 and 100 °C) significantly increased the rate of germination relative to controls. Cytisus scoparius is more favoured by fire action than Genista florida, with germination rates slightly greater following 100 °C for 5 min and 130 °C for 1 min than after mechanical scarification.     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick