Acid Fuchsin as a Stain—A Refinement in Manufacture

From the study of 38 samples of acid fuchsin prepared from several types of basic fuchsin and under varying conditions it is found that rosanilin sulfonated between 80° and 85°C. gives the best results in the Van Gieson staining technic. Staining tests also show that a satisfactory acid fuchsin will give the best results when employed with picric acid in the ratio of 1 part of the 1 per cent aqueous acid fuchsin to 20 parts of the aqueous picric acid. Details for the preparation and use of acid fuchsin are given.     

Acid Fuchsin as a Connective Tissue Stain After Phosphomolybdotungstic Mordanting

Certain acid fuchsias stain connective tissue deep red after phosphomolybdotungstic mordanting in a modified Masson procedure, others are entirely unsatisfactory for mis purpose. Spectrophotometric examination gives no reliable criteria for separation of acid fuchsins satisfactory for this purpose from unsatisfactory ones. Sulphonation of basic fuchsin with 3.5 to 4 parts of 25-30% fuming H₂SO₄ to 1 part of dye gives a satisfactory product at temperatures as low as 65 to 70°C. in 30 minutes, while use of 5 to 7.5 parts of acid at this and at higher and lower temperatures gives unsatisfactory products. Satisfactory products may be produced with 15% fuming H₂SO₄ in similar quantities, and even with concentrated H₂SO₄, but some unconverted basic fuchsin remains with both and, with the latter, lower quantities give unsatisfactory products. Brief chemical studies indicate that oversulphonation may occur in the manufacture of acid fuchsin and that this is just as deleterious as undersulphonation.     

A Simple Toluidine Blue—Basic Fuchsin Stain for Spermatozoa in Epoxy Sections

Differential staining of cell components of spermatozoa is readily accomplished in Epon or Araldite sections 0.5-1 μ thick from rat and hamster testis and epididymis, and stained as follows: 1% aqueous toluidine blue buffered at pH 6, 0.5-3 min at 90 C; washed in distilled water; 1% basic fuchsin in 50% alcohol, 3-5 min at 20-25 C; differentiated with 70% alcohol; allowed to dry; and mounted in a resin of high refraction (DPX was used). Results: acrosome, bright magenta; nucleus, deep blue; mitochondrial sheath of the middle-piece, pinkish purple; and tail, pale red. This procedure combined with staining of collagen by applying 2% aqueous phosphotungstic acid 1-2 min as a mordant, followed by 1% light green in 50% alcohol containing 1% acetic acid, 1-2 min at 20-25 C, gives polychromatic staining and is useful as a general stain for other epoxy-embedded tissues.     

Basic Fuchsin as A Nuclear Stain

Five distinct nuclear stains and staining procedures which utilize basic fuchsin as the dye have been studied, compared and tested on a Feulgen-weak fungus, Blastomyces dermatitidis, and other fungi.

Aqueous basic fuchsin has been shown to be an excellent, though impermanent, stain with which to study the nuclei of this and other fungi. The conditions under which formaldehyde acts as a mordant for basic fuchsin and produces a permanent nuclear stain have been established.

Comparison of crystal violet and basic fuchsin suggests that the mordanting action of the aldehyde operates through the para-amino groups of the dye. Certain other basic dyes were not mordanted by formaldehyde.

Gentle acid hydrolysis of the tissues has been found to be essential both to the specificity of the dye as a nuclear stain and to the mordanting effect of the aldehyde.

The possible relationship of these observations to the Feulgen reaction is discussed. A protocol for the method developed is presented.     

Carbol Fuchsin as A Stain for Human Chromosomes

Cells derived from cultures of bone marrow or leucocytes were treated with hypotonic citrate solution, squashed in 45% acetic acid frozen with CO₂ to allow removal of the cover glass without disturbing the smear, and stained by the following schedule: absolute alcohol, 5 min; coat with 0.2% parlodion and air dry; 70% alcohol, 5 min; distilled water, 5 min; stain 2-5 min in a mixture of 45 ml of a 0.3% solution of basic fuchsin in 5% phenol, 6 ml of glacial acetic acid, and 6 ml of 37% formaldehyde. Differentiate and dehydrate in absolute alcohol, clear in xylene and cover. The stain is durable for several weeks if slides are stored in darkness when not in use. Results resemble those obtained by Feulgen or aceto-orcein methods.     

Basic Fuchsin in Acid Alcohol: A Simplified Alternative to Schiff Reagent

The use of Schiff reagent to demonstrate polysaccharides (after prior periodic oxidation) and nucleic acids (after prior acid hydrolysis) is unnecessary since the same results are obtained by substituting a 20 min staining in a 0.5% w/v solution of basic fuchsin in acid alcohol (ethanol-water-concentrated HC1, 80:20:1) followed by a rinse in alcohol. The shade of the basic fuchsin staining is a little yellower than that achieved with Schiff reagent but the selectivity, light fastness, response to different fixatives, and to prior histo-chemical blocking of the tissue section were much the same for the two methods. The need for prior oxidation or hydrolysis and the inhibitory effect of aldehyde blocking techniques indicate that basic fuchsin, like Schiff reagent, reacts with aldehyde groups. Infrared studies indicate that for cellulose the reaction product is an azomethine.     

Basic Fuchsin as an Indicator in Endo's Medium

The writer has made an investigation of various samples of basic fuchsin for use in the Endo medium for differentiating the bacteria of the colon-typhoid group. Various different concentrations of the fuchsin samples have been used in making the media. The conclusions are as follows:

American made fuchsins differ markedly in their alcohol solubility properties. They contain materials which are very readily soluble in 95% alcohol, but which are precipitated by sodium sulphite.

This precipitation may be prevented by increasing the dilution of the fuchsin in alcohol.

In order to secure more dependable results in the use of decolorized basic fuchsin as an indicator in Endo Agar, it is advisable to test the fuchsin in different dilutions in alcohol in order to secure a completely decolorized solution. It is also advisable to carefully test those fuchsins which decolorize only in high dilutions with a known organism in Endo agar before relying on it as a satisfactory indicator for the presence of sewage organisms.     

A Methylene Blue-Basic Fuchsin Stain for Mast Cells in Paraffin Sections

In paraffin sections of rat tissue it is possible to stain mast cell granules blue in contrast to red nuclei, pale blue cytoplasmic ribonucleic acid, and colorless collagen. This is done by the following mixture: 1% methylene blue (pure, not polychrome), 9 ml; 0.1% basic fuchsin, 9 ml; glacial acetic acid, 2 ml. Stain formol-fixed, paraffin-processed sections for 5 min, wash in water and pass through acetone, 2 changes, 10 sec total, to xylene and a polystyrene mounting medium.     

RADseq as a valuable tool for plants with large genomes—A case study in cycads

Full genome sequencing of organisms with large and complex genomes is intractable and cost ineffective under most research budgets. Cycads (Cycadales) represent one of the oldest lineages of the extant seed plants and, partly due to their age, have incredibly large genomes up to ~60 Gbp. Restriction site‐associated DNA sequencing (RADseq) offers an approach to find genome‐wide informative markers and has proven to be effective with both model and nonmodel organisms. We tested the application of RADseq using ezRAD across all 10 genera of the Cycadales including an example data set of Cycas calcicola representing 72 samples from natural populations. Using previously available plastid and mitochondrial genomes as references, reads were mapped recovering plastid and mitochondrial genome regions and nuclear markers for all of the genera. De novo assembly generated up to 138,407 high‐depth clusters and up to 1,705 phylogenetically informative loci for the genera, and 4,421 loci for the example assembly of C. calcicola. The number of loci recovered by de novo assembly was lower than previous RADseq studies, yet still sufficient for downstream analysis. However, the number of markers could be increased by relaxing our assembly parameters, especially for the C. calcicola data set. Our results demonstrate the successful application of RADseq across the Cycadales to generate a large number of markers for all genomic compartments, despite the large number of plastids present in a typical plant cell. Our modified protocol was adapted to be applied to cycads and other organisms with large genomes to yield many informative genome‐wide markers.     

A Refinement in Mechanized Plating

A plating instrument is described for use in the determination of viable counts of micro-organisms in foods and other biological materials. It is based on the linear distribution of a fixed volume of inoculum on the surface of a rotating agar plate in a continuous spiral fashion. The rate of inoculation is controlled so that from the relative position and number of the emerging colonies, viable counts up to 10⁹/ml can be obtained without the necessity to dilute the sample. Counting can be rapid and the time taken for reading the plates can be reduced considerably. The instrument includes controls for the variability in the agar thickness, uneven surface as the result of drying, multi-plate inoculation without re-charging and speedy de-contamination between inoculations.     

The tonoplast proton—Translocating ATPase of higher plants as a third class of proton—Pumps

Taken together, all the data reported recently in the literature suggest that tonoplast ATPase belongs to a new class of proton pumps. To date, the most studied system is the proton-pumping ATPase from the tonoplast of Hevea latex. Its main characteristics are presented. It resembles the mitochondrial ATPase in its specificity, its substrate affinity, and its sensitivity to different inhibitors. However, for some aspects, it resembles the plasma membrane system in its response to other inhibitors tested (quercetin for example). It differs from both ATPases in its sensitivity to nitrate as well as by its molecular structure, i.e. a complex exhibiting a least 4 or 5 polypeptides. These results favor the existence of a third class of proton pumps, intermediate between the F1F0-class and the E1E2-class.     

Wax as a Mechanism for Protection against Photoinhibition — A Study of Cotyledon orbiculata

The leaves of the Crassulacean acid metabolism plant Cotyledon orbiculata have a waxy coating which is highly reflective but can be easily removed by brushing. This provided an ideal system in which to investigate the role of epidermal wax as a possible photoprotectant. Removal of the wax, prior to exposure to natural sunlight, resulted in substantial decreases in F_v/F_m and in severe cases evidence of photoinhibitory damage, as indicated by a rise in F_o. Leaves from which wax had been removed also showed higher conversion of violaxanthin to zeaxanthin than waxed leaves. Recovery of brushed leaves over a 12 day period was correlated with an increase in the total pool of xanthophyll cycle components. This study suggests that the presence of highly reflective wax on the epidermis may confer significant photoprotection to plants exposed to high solar radiation environments.     

A mechanistic study of the histochemical reactions between aldehydes and Basic Fuchsin in acid alcohol used as a simplified substitute for Schiff's reagent

Synopsis For the identification of polysaccharides after periodic acid oxidation or of DNA after acid hydrolysis, a solution of 0.5% w/v Basic Fuchsin in acid alcohol (water-ethanol-concentrated hydrochloric acid 80:20:1 by volume) may be used instead of Schiff's reagent. Sections are stained in the Fuchsin solution for 20 min, after which the unreacted dye is washed off with ethanol. Except for its yellower colour the Fuchsin staining is almost indistinguishable from Schiff's reagent staining.Histochemical blocking studies indicated that the Fuchsin stain, like Schiff's reagent, reacts with aldehyde groups or subsequent oxidation products. The results of studies of model systems (cellulose film oxidized by periodic acid and also of aqueous formaldehyde solution) in which infra-red spectroscopy and, where appropriate, chromatography were used are consistent with the initial coloured products being azomethines which may react further to produce coloured secondary amine derivatives.     

Fuchsin fluorescence in Mycobacterium tuberculosis: the Ziehl-Neelsen stain in a new light

Tuberculosis (TB) is often diagnosed by observation of reddish pink fuchsin-stained Mycobacterium tuberculosis (MTB) in Ziehl-Neelsen (Z-N) stained smears by transmitted light microscopy. MTB too faintly stained with fuchsin to be seen by transmitted light may be detected by their green-excited orange-red fluorescence; this finding may be clinically relevant.     

A review of wheat diseases—a field perspective

Wheat is one of the primary staple foods throughout the planet. Significant yield gains in wheat production over the past 40 years have resulted in a steady balance of supply versus demand. However, predicted global population growth rates and dietary changes mean that substantial yield gains over the next several decades will be needed to meet this escalating demand. A key component to meeting this challenge is better management of fungal incited diseases, which can be responsible for 15%–20% yield losses per annum. Prominent diseases of wheat that currently contribute to these losses include the rusts, blotches and head blight/scab. Other recently emerged or relatively unnoticed diseases, such as wheat blast and spot blotch, respectively, also threaten grain production. This review seeks to provide an overview of the impact, distribution and management strategies of these diseases. In addition, the biology of the pathogens and the molecular basis of their interaction with wheat are discussed.     

Biogenic calcite particles from microalgae—Coccoliths as a potential raw material

Synthetic calcite (CaCO₃) particles are found in a broad range of applications. The geometry of particles produced from limestone or precipitation are versatile but limited to basic shapes. The microalga Emiliania huxleyi produces micro‐structured calcite platelets, called coccoliths. This article presents the results of an application‐orientated study, which includes characteristic values also used in the calcite industry for particle evaluation. It is demonstrated that coccoliths are significantly different from all industrial particles produced so far. Coccoliths are porous particles, mainly consisted of calcium carbonate, with further elements such as Mg, Si, Sr, and Fe often embedded in their structure. Their structure is extremely sophisticated, while the overall particle morphology and particle size distribution are homogeneous. This study gives a first inside into the potential of these exceptional objects and may set further impulses for their utilization in specific calcite particle applications.