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.     

Some Synthetic Resins IN Combined Fixing, Staining and Mounting Media

Many of the recently devised plasticizers and resins can be utilized to advantage in cytological technics. Some of them have solubilities which enable us to incorporate them in such fixing and staining solutions as aceto-carmine and propiorric-carmine. They are non-volatile, do not alter the fixation images of the fluids with which they are mixed, and serve as mounting media as the volatile components evaporate. Thus it is possible to make a permanent slide in a single operation. These newer compounds are better adapted for this technic than are the natural balsams which have been used previously, as their greater tolerance for water provides a much greater margin of safety. Procedures are described for the utilization of (1) Rezyl 7020, a water-soluble resin (now, unfortunately, not available), which dries to form a water insoluble film, (2) Amberol 750 and (3) Bakelite BR-7160, two alcohol soluble resins, more miscible in solutions containing water than are the natural balsams. Formaldehyde can be included in the aceto-carmine and propionic-carmine fluids with the result that more nuclear detail is preserved. Lacto-gelatin has some valuable properties as a mounting medium and can be used when the specimen is stained with orcein. Carmine, which gives a permanent stain in Rezyl 7020, Amberol 750 and Bakelite BR-7160 fades in lacto-gelatin.     

On the Composition of “Viscol” and the Employment of Gum Arabic for Mounting Media

“Viscol”, a water soluble permanent mounting medium hardening through evaporation of water under the cover glass, has been analyzed. It proves to consist of a mixture of gum arabic, glycerol, phenol and water and is especially suitable for simple botanical preparations. The use of gum arabic for hardening permanent mounting media is reviewed. Instead of a glycerol-phenol-water mixture a lactophenol, potassium acetate or zinc chloride solution mixed with gum arabic may be used for a permanent mounting medium. However, gum arabic contains calcium, magnesium and potassium ions giving crystals with the solutions mentioned. In the case of lactophenol and potassium acetate, the calcium and magnesium ions must be removed beforehand, which is done by precipitation with sodium or potassium carbonate. In the case of zinc chloride the potassium ions must be removed, which is done by dialysis with zinc chloride. It is pointed out that the same principles may be used for a great variety of different mounting media.     

Polarization microscopy and microspectrophotometry of Sirius Red, Picrosirius and Chlorantine Fast Red aggregates and of their complexes with collagen

Summary A detailed quantitative analysis of the anisotropic properties of Sirius Red F3B, Picrosirius, and Chlorantine Fast Red crystals, and of their complexes with a macromolecularly oriented protein either in a pure form or as part of a tissue structure was carried out. Collagen I was used as the protein model. Linear dichroism and dispersion of birefringence were investigated in dye aggregates, in stained filaments of collagen I and in collagen bundles in sections of tendon. A positive linear dichroism, the characteristics of which varied as a function of the dye type used, was demonstrated for the dye aggregates and stained substrates. However, even thin regions of the stained tendon collagen bundles showed very high absorbances, differing from the pattern reported previously, for collagen stained with another sulphonated azo dye, Xylidine Ponceau. Consequently, not all these dyes enable protein concentration and orientation to be determined in collagen-containing structures. From the linear dichroism patterns it is assumed that the long axis of the molecules of these azo dye is mostly parallel to that of filaments of pure collagen I and statistically parallel to the long axis of collagen bundles of tendon sections. The dye aggregates and, stained pure collagen I and tendon collagen bundles exhibited birefringent images with interference colours that varied as a function of thickness and packing state of the preparations, which is in agreement with reports in the literature. The optical retardations of the collagen bundles increased by a factor of 5–6 times after staining with Picrosirius. From data on form dichroism it is concluded that when studying the macromolecular orientation of collagen preparations stained with azo dyes, the choice of the mounting medium deserves consideration.     

Vinylpyrrolidone-Vinyl Acetate Copolymers as Mounting Media for Azo and Other Dyes

Vinylpyrrolidone-vinyl acetate copolymers (Antara Chemicals, General Aniline and Film Corp., New York N. Y., PVP/VA, E-735 and E-635) were employed as mounting media by adition of ethanol-water to the concentrated ethanolic solution of these plastics (25% plastic in 50% ethanol). The E-735 copolymer was frequently employed and is specifically recommended because it exhibits the highest degree of water tolerance. This type of mounting medium was found to be especially satisfactory in the preservation of azo, oxidation, and other histochemically derived dyes. The medium is useful also in the preservation of stains for fat and certain metachromatic dyes. The inexpensive nature of this mountant and its ease of application recommend it as a useful substitute for glycerol-gelatin.     

Characterization of azo reduction activity in a novel ascomycete yeast strain

Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity of I. occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.     

Characterization of Azo Reduction Activity in a Novel Ascomycete Yeast Strain

Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity of I. occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.     

Assessment of the effect of azo dye RP2B on the growth of a nitrogen fixing cyanobacterium--Anabaena sp

Certain nitrogen fixing cyanobacteria are diazotrophic, which profoundly impacts the aquatic ecosystem chemically and biologically. Although certain types are banned due to their carcinogenicity, azo dyes are commonly used in the dyeing or textile industry. This work investigates the effect of azo dye on the growth of cyanobacteria. Anabaena sp. isolated from the Da Jia Brook is an odor producing, nitrogen fixing cyanobacterium. The growth rates of Anabaena sp. in the media with or without nitrogen source were 3.56 x 10(-2) mg/ml day and 2.44 x 10(-2) mg/ml day, respectively. Anabaena sp. could not use azo dye RP2B as the nitrogen source. Experimental results indicated that the growth of Anabaena sp. was inhibited in the medium containing RP2B. The degree of inhibition increased from 50% to 81% with an increasing concentration of RP2B (0-50 mg/l). The IC-50 (inhibitory concentration) of RP2B on the growth of Anabaena sp. was 5 mg/l (as based on dry weight) or 7 mg/l (as measured by chlorophyll a).     

Effect of inducers on the decolorization and biodegradation of textile azo dye Navy blue 2GL by Bacillus sp. VUS

Bacillus sp. VUS decolorized azo dye Navy blue 2GL in 48 h at static anoxic condition in yeast extract medium, whereas it took only 18 h for the decolorization in presence of CaCl₂. Different inducers played role in the decolorization of Navy blue 2GL. CaCl₂ found to be the most effective inducer among all inducers tested. The activity of enzymes like lignin peroxidase, laccase and reductases viz. NADH-DCIP, azo and riboflavin induced during decolorization represents their role in the biodegradation. Extracellular LiP and intracellular laccase activity induced with CaCl₂. Yeast extract was best medium for faster decolorization than other media. UV–vis spectrophotometer analysis and visual examinations showed decolorization of dye. High performance liquid chromatography, Fourier transforms infrared spectroscopy showed degradation of dye. Gas Chromatography-Mass Spectroscopy revealed formation of 4-Amino-3-(2-bromo-4, 6-dinitro-phenylazo)-phenol and acetic acid 2-(-acetoxy-ethylamino)-ethyl ester as final products. Bacillus sp. VUS also decolorized synthetic effluent. Phytotoxicity study showed detoxification of Navy blue 2GL.     

脱色希瓦氏菌(Shewanella decolorationis)S12T的脱色特性

从印染废水活性污泥中分离到一株高效染料脱色菌,经鉴定该菌株为希瓦氏菌属的一个新种,命名为脱色希瓦氏菌(Shewanelladecolorationis)S12T。该菌株在偶氮染料浓度为50mg/L的培养基中培养4h后,染料去除率达到96%,对偶氮染料的最高脱色浓度达到2000mg/L。在浓度为500mg/L的偶氮染料平板上生长4d后,可观察到明显的脱色圈。全波长光谱扫描的结果表明希瓦氏菌S12T以生物降解的方式对偶氮染料进行脱色。希瓦氏菌S12T的脱色酶为组成型的胞内酶。     

Mechanism of enzymatic degradation of the azo dye Orange II determined by ex situ 1H nuclear magnetic resonance and electrospray ionization-ion trap mass spectrometry

Removal of azo dye effluents generated by textile photography industries is a main issue in wastewater treatment. Enzymatic treatment of dyes appears to be one of the most efficient processes for their degradation. The elucidation of degradation pathways is of special interest considering health and environmental priorities. Ex situ nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization (ESI)-ion trap mass spectrometry performed directly on incubation medium have been used for the first time to follow kinetics of sulfonated azo dye Orange II enzymatic degradation. Nine transformation products were identified using these complementary analyses performed ex situ without any prior treatment. Three types of cleavage are proposed for the degradation pathway: (i) a symmetrical splitting of the azo linkage that leads to the formation of 4-aminobenzenesulfonate (and 1-amino-2-naphthol, not detected); (ii) an asymmetrical cleavage on the naphthalene side that generates 1,2-naphthoquinone and 4-diazoniumbenzenesulfonate as products, with the latter one being transformed into 4-hydroxybenzensulfonate; and (iii) a third degradation pathway that leads to 2-naphthol and 4-hydroxybenzenesulfonate. Moreover, three other intermediates have been identified. This study, which constitutes the first concomitant use of (1)H NMR spectroscopy and ESI-ion trap mass spectrometry in this field, illustrates the indubitable interest of the ex situ approach.     

Modified Procedure for the Microscopic and Macroscopic Study of Viral Plaques

A virus plaque method that consistently gives good visual contrast for macroscopic observation and also permits microscopic study is described. Cells are grown in plastic flasks and the gelled overlay medium can be of any desired agar concentration or volume. A fixing solution is used prior to removal of the agar overlay, and dye is added to the fixing solution or staining can follow fixation and agar removal. The bottom of the flask with the fixed monolayer is separated from the rest of the container and handled as a slide. A new mounting medium is described.     

Advantages of Epoxy Resin as a Mounting Medium for Light Microscopy

The need for very durable mounting is especially felt in the teaching of parasitology and mycology; otherwise, the availability of microscope slides may depend on the use of fresh specimens. Resinous mounts and those in aqueous media sealed with fingernail lacquer, paraffin or asphalt do not preserve specimens satisfactorily. Polyvinylpyrrolidone (pvp), a water-soluble mounting medium described by Burstone (1962), cannot be applied directly for mounting of insects and certain other parasites which have water-repelling integuments; moreover, pvp bleaches eosin. Grimley et al. (1965) prepared large epoxy sections of tissues from which areas for electron microscopy could be selected. This procedure however is designed for electron microscopic techniques whereas the present paper describes a direct epoxy mounting method to produce permanent mounts for light microscopy.     

Simplified Permanent Aceto-Carmine Smears with A Water-Miscible Mountant

By employing a water-miscible mountant with a good refractive index, permanent aceto-carmine smears can be made from any of a variety of killing, fixing, and staining schedules. Clear-col, a commercial preparation, orginally developed for mounting fungi from various water-containing media, is employed for anther smears by placing the medium directly on the final stage of aceto-carmine staining schedule. Thus, no dehydration and clearing are necessary. By controlling amounts of acetic acid either mixed with Clearcol or on smears, clear, well-stained preparations can be produced.     

Decolorization of orange G by<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> monokaryotic isolates with different laccase activity

The effect of enhanced laccase (Lac) activity (obtained after copper addition to cultivation media) on decolorization of azo dye Orange G in two basidiospore-derived monokaryotic isolates of Pleurotus ostreatus was determined. The high Lac-producing isolate efficiently decolorized Orange G. The low-producing isolate showed only poor decolorization ability during cultivation in liquid medium and no decolorization on agar plates containing Orange G after a 25-d growth. A substantial enhancement of Lac activity caused by copper addition into cultivation media was detected in both isolates but, at the same time, the biomass production decreased and decolorization rate was reduced.     

Non-aqueous permanent mounting for immunofluorescence microscopy

It is generally assumed that an aqueous mounting medium is necessary for the preservation of immunofluorescent-labelled microscopical preparations and polyvinyl alcohol-based solutions (e.g. Mowiol) being the most frequently used mounting media; however, both the quality and intensity of the fluorescence signal in most immunolabelled preparations after aqueous mounting slowly diminish with time, and finally, samples become unsuitable for examination. In the present work, we describe a very simple and rapid non-aqueous mounting procedure for cultured cells and tissue sections, which preserves the fluorescent signal in an excellent way after immunodetection or use of other specific labelling methods. It is based on the current histological protocol in which, after fluorescence labelling, preparations are dehydrated in ethanol, cleared in xylene and mounted in DePeX. Using this non-aqueous mounting medium, the fluorescent signal remains high and stable, allowing a suitable and permanent preservation of labelled and counterstained microscopical preparations.     

Azo dye decolorization with a mutant Escherichia coli strain

A recombinant Escherichia coli strain (E. coli NO3) containing genomic DNA fragments from azo-reducing wild-type Pseudomonas luteola strain decolorized a reactive azo dye (C.I. Reactive Red 22) at approx. 17 mg dye h^–1 g cell. The ability to decolorize the azo dye probably did not originate from the plasmid DNA. Acclimation in azo-dye-containing media gave a nearly 10% increase in the decolorization rate of E. coli NO3. Growth with 1.25 g glucose l^–1 completely stopped the decolorization activity. When the decolorization metabolites from E. coli NO3 were analyzed by HPLC and MS, the results suggested that decolorization of the azo dye may be due to cleavage of the azo bond.     

Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX

Sphingomonas sp strain 1CX was isolated from a wastewater treatment plant and is capable of aerobically degrading a suite of azo dyes, using them as a sole source of carbon and nitrogen. All azo dyes known to be decolorized by strain 1CX (Orange II, Acid Orange 8, Acid Orange 10, Acid Red 4, and Acid Red 88) have in their structure either 1-amino-2-naphthol or 2-amino-1-naphthol. In addition, an analysis of the structures of the dyes degraded suggests that there are certain positions and types of substituents on the azo dye which determine if degradation will occur. Growth and dye decolorization occurs only aerobically and does not occur under fermentative or denitrification conditions. The mechanism by which 1CX decolorizes azo dyes appears to be through reductive cleavage of the azo bond. In the case of Orange II, the initial degradation products were sulfanilic acid and 1-amino-2-naphthol. Sulfanilic acid, however, was not used by 1CX as a growth substrate. The addition of glucose or inorganic nitrogen inhibited growth and decoloration of azo dyes by 1CX. Attempts to grow the organism on chemically defined media containing several different amino acids and sugars as sources of nitrogen and carbon were not successful. Phylogenetic analysis of Sphingomonas sp strain 1CX shows it to be related to, but distinct from, other azo dye-decolorizing Sphingomonas spp strains isolated previously from the same wastewater treatment facility. Received 19 May 1999/ Accepted in revised form 11 August 1999     

Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium.

Under anaerobic conditions the sulfonated azo dye Mordant Yellow 3 was reduced by the biomass of a bacterial consortium grown aerobically with 6-aminonaphthalene-2-sulfonic acid. Stoichiometric amounts of the aromatic amines 6-aminonaphthalene-2-sulfonate and 5-aminosalicylate were generated and excreted into the medium. After re-aeration of the culture, these amines were mineralized by different members of the bacterial culture. Thus, total degradation of a sulfonated azo dye was achieved by using an alternating anaerobic-aerobic treatment. The ability of the mixed bacterial culture to reduce the azo dye was correlated with the presence of strain BN6, which possessed the ability to oxidize various naphthalenesulfonic acids. It is suggested that strain BN6 has a transport system for naphthalenesulfonic acids which also catalyzes uptake of sulfonated azo dyes. These dyes are then gratuitously reduced in the cytoplasm by unspecific reductases.     

Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium.

Under anaerobic conditions the sulfonated azo dye Mordant Yellow 3 was reduced by the biomass of a bacterial consortium grown aerobically with 6-aminonaphthalene-2-sulfonic acid. Stoichiometric amounts of the aromatic amines 6-aminonaphthalene-2-sulfonate and 5-aminosalicylate were generated and excreted into the medium. After re-aeration of the culture, these amines were mineralized by different members of the bacterial culture. Thus, total degradation of a sulfonated azo dye was achieved by using an alternating anaerobic-aerobic treatment. The ability of the mixed bacterial culture to reduce the azo dye was correlated with the presence of strain BN6, which possessed the ability to oxidize various naphthalenesulfonic acids. It is suggested that strain BN6 has a transport system for naphthalenesulfonic acids which also catalyzes uptake of sulfonated azo dyes. These dyes are then gratuitously reduced in the cytoplasm by unspecific reductases.