Lacto-Phenol Preparations

More or less permanent mounts of fungi, algae, root tips, epidermis, germinating spores, and other small objects may be made readily by transferring the material to Amann's lacto-phenol containing anilin blue, W. S. or acid fuchsin, used singly or mixed. The addition of 20 to 25% of glacial acetic acid to these mixtures is frequently advantageous; or material may be stained with various dyes—acid fuchsin, anilin blue, W. S. (cotton blue), rose bengal, phloxine, hematoxylin—in aqueous solutions containing 5% of phenol, and then mounted in lacto-phenol, 50% glycerin or phenolglycerin, depending on the dye used. The phenol solutions of acid fuchsin and anilin blue are acidified with acetic acid and those of rose bengal and phloxine are made slightly alkaline with ammonium hydroxide. The addition of ferric chloride to acid fuchsin or acidified hematoxylin may improve staining. Fixation may be preferable but may be omitted, especially with fungi. Formulae for the mounting media and ten staining mixtures are given.     

Further Experiments with the Masson Trichrome Modification of Mallory's Connective Tissue Stain

Several dyes, notably ponceau 2R, azofuchsin 3B, nitrazine yellow, and Biebrich scarlet may replace imported “ponceau de xylidin” in the Masson ponceau acid fuchsin mixture. Of these Biebrich scarlet appears to be the best and may be used without acid fuchsin.

A mixture of equal parts of 5% solutions of phosphomolybdic and phosphotungstic acids is much superior to either acid alone and gives adequate mordanting in 1 minute at 22°C.

With the fast green modification, times in plasma and fiber stains can be reduced to 2 minutes each. With anilin blue a 4-minute plasma stain is required. One-minute final differentiation in 1% acetic acid is adequate.

Primary mordanting of formalin material may be accomplished by 5 minutes in saturated aqueous mercuric chloride or 2 minutes in saturated alcoholic picric acid. Three minutes washing in running water is required after these mordants.     

Methods for the Standardization of Biological Stains: Part IV

In this paper are given methods for determining the suitability of certain dyes of the triphenylmethane group for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission, in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Malachite green, brilliant green, light green SF yellowish, fast green FCF, basic fuchsin (rosanilin and pararosanilin), acid fuchsia, methyl violet, crystal violet, gentian violet, methyl green and anilin blue. For each of these dyes, methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

The Acidophilia of Elastin

Conditions relevant to the acceptance of acid dyes by the elastin of the aorta are as follows: (a) Fixation. Cold alcohol is poor, formol-sublimate, hest; others are intermediate. (h) Acid dyes used. Bromphenol blue is best, light green is poor, acid fuchsin is unstable in alkaline solution. Ponceau, aniline blue, fast green, chromotrope, erythrosin, and eosin are only moderately useful. (c) Staining pH. Elastica shares with eosinophil granules the property of retaining acid dye up to pH 11. (d) Autolysis. There is no effect even after 24-36 hr post mortem on acid dye affinity. (e) Speed of staining. Minor variations will appear if staining is less than 30 min. (f) Species of animal. Rat, mouse, cat, sheep, rabbit, guinea pig, opossum, ferret, rhesus monkey, horse, cow, and pig all possess acidophilic aortic elastin. A special feature has been the effect of age on the staining of human aortic elastin; bromphenol blue washes out quickly from the aortic elastin of old subjects, slowly from that of infants.     

A Modification of the Technic of Handling Chick Embryos

In this paper are given methods for determining the suitability of certain dyes of the triphenylmethane group for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission, in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Malachite green, brilliant green, light green SF yellowish, fast green FCF, basic fuchsin (rosanilin and pararosanilin), acid fuchsia, methyl violet, crystal violet, gentian violet, methyl green and anilin blue. For each of these dyes, methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

The Acidophilia of Elastin

Conditions relevant to the acceptance of acid dyes by the elastin of the aorta are as follows: (a) Fixation. Cold alcohol is poor, formol-sublimate, hest; others are intermediate. (h) Acid dyes used. Bromphenol blue is best, light green is poor, acid fuchsin is unstable in alkaline solution. Ponceau, aniline blue, fast green, chromotrope, erythrosin, and eosin are only moderately useful. (c) Staining pH. Elastica shares with eosinophil granules the property of retaining acid dye up to pH 11. (d) Autolysis. There is no effect even after 24-36 hr post mortem on acid dye affinity. (e) Speed of staining. Minor variations will appear if staining is less than 30 min. (f) Species of animal. Rat, mouse, cat, sheep, rabbit, guinea pig, opossum, ferret, rhesus monkey, horse, cow, and pig all possess acidophilic aortic elastin. A special feature has been the effect of age on the staining of human aortic elastin; bromphenol blue washes out quickly from the aortic elastin of old subjects, slowly from that of infants.     

A method for preparing and staining histological sections containing titanium implants for light microscopy

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 microns thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.     

A Method for Preparing and Staining Histological Sections Containing Titanium Implants for Light Microscopy

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 μm thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.     

A new fluorescent staining method for callose of microspore mother cells during meiosis

To observe the dynamic behavior of callose of microspore mother cells during meiosis, we developed a convenient, rapid and efficient staining method using an improved carbol fuchsin/aniline blue solution. The stained microspore mother cells during meiosis showed yellowish green callose, red cytoplasm and dark red chromosomes when excited with blue light, which produced a contrasting image with a three-dimensional effect. When stained with only improved carbol fuchsin solution, the cells had red cytoplasm and chromosomes when excited with green light. The improved carbol fuchsin solution can be used to replace other more expensive DNA-specific dyes, such as DAPI and H33258, to reduce experimental costs.     

A new fluorescent staining method for callose of microspore mother cells during meiosis

To observe the dynamic behavior of callose of microspore mother cells during meiosis, we developed a convenient, rapid and efficient staining method using an improved carbol fuchsin/aniline blue solution. The stained microspore mother cells during meiosis showed yellowish green callose, red cytoplasm and dark red chromosomes when excited with blue light, which produced a contrasting image with a three-dimensional effect. When stained with only improved carbol fuchsin solution, the cells had red cytoplasm and chromosomes when excited with green light. The improved carbol fuchsin solution can be used to replace other more expensive DNA-specific dyes, such as DAPI and H33258, to reduce experimental costs.     

Method of counterstaining preparations for immunofluorescent studies of the pituitary]

To elucidate nonfluorescent structural elements of the hypophyseal parenchyma for immunofluorescent investigations, properties of some dyes most commonly applied for hypophysis staining have been studied. Such dyes as paraldehide-fuchsin, light green, orange G, chromotrop 2R, hematoxylin, eosin, fuchsin, azocarmin possess their own intensive luminescence and block immunofluorescence completely. Some other dyes (trypan blue, bromthymol blue, aniline blue, malachite green, methyl green) though not blocking immunofluorescence, they do not reveal hypophyseal cellular elements distinctly enough. Good results have been obtained with 0.3% water solution of toluidine blue, 0.5% solution of methylene light blue, methylene blue, as well as with Gram--Weigert's staining and with gallocyanin after Einarson. For special staining of corticotropocytes, the authors recommend 0.1% solution of bromphenol blue in barate buffer, pH 8.2.     

Comparison of historic Grübler dyes with modern counterparts.

Seventeen Grübler dyes produced in Germany between 1880 and 1939 were examined in this study. These dyes were: fuchsin-bacillus, diamond fuchsin, fuchsin S acid, rubin S, safranin O water soluble, safranin yellowish water soluble, methyl eosin, Sudan III, scarlet R, auramine, orange G, aniline blue, pyronin, carmine, lithium carmine, hematein and aurantia. Spectrophotometry and staining characteristics were used to determine the maximum absorbance and efficacy of each dye in common staining techniques. The spectral curves and staining characteristics of these dyes compared well with modern dyes used as controls. Fuchsin bacillus and diamond fuchsin are synonyms for basic fuchsin. Fuchsin S acid and rubin S are synonyms for acid fuchsin. The scarlet R sample was the same as the Sudan III. The two safranins were the same. The basic fuchsin samples were unsuitable for preparation of Schiff's reagent. Both basic fuchsin and pyronin samples were less concentrated than modern counterparts. It is noteworthy that the dyes worked well after up to 100 years in storage, and this observation indicates that dyes can have a long shelf life when stored in cool, dry, air-tight conditions.     

A Spectrophotometry Analysis of Tissue Staining

By comparing spectral absorption curves of representative staining solutions and of substances stained with these solutions it is shown that information may be obtained regarding chemical changes associated with the staining process. The stains used in these determinations were acid fuchsin, anilin blue, azo-carmine G, basic fuchsin, eosin Y, orange G, picric acid and Sudan IV. The substrates stained were gelatin, tendon, blood plasma, thymus gland and fat.

Aqueous basic fuchsin and fuchsin-sulfurous reagent to which formalin was added (Setoff reaction) are different stains. The spectral absorption curves for staining solutions and substances stained with the solutions were comparable. Within the limitations of the spectrophotometry methods and stains employed, there was no evidence of significant chemical alteration in the chromophore radicals of the stains associated with the process of tissue staining.     

A Method for the Staining of Bacterial Flagella

A modification of the Loeffler method of staining bacterial flagella is proposed. The chief points of the modification are: The cultures are inoculated into distilled water after two successive daily transfers on agar slants, and the distilled water cultures are incubated at optimum temperature for from 48 to 72 hours. The mordant (tannic acid, ferrous sulphate, basic fuchsin) is allowed to stand 18 to 24 hours before use, and then cleared by centrifuging or filtering. An anilin water fuchsin is used as a stain. No heat is used for either mordanting or staining; but both mordant and stain are allowed to act on the preparation for 15 minutes. The writer finds the method admirably adapted for use in class work, where nearly 100 per cent success has been obtained except in the case of some three or four species of bacteria that are especially difficult to stain.     

A Method for the Staining of Bacterial Flagella

A modification of the Loeffler method of staining bacterial flagella is proposed. The chief points of the modification are: The cultures are inoculated into distilled water after two successive daily transfers on agar slants, and the distilled water cultures are incubated at optimum temperature for from 48 to 72 hours. The mordant (tannic acid, ferrous sulphate, basic fuchsin) is allowed to stand 18 to 24 hours before use, and then cleared by centrifuging or filtering. An anilin water fuchsin is used as a stain. No heat is used for either mordanting or staining; but both mordant and stain are allowed to act on the preparation for 15 minutes. The writer finds the method admirably adapted for use in class work, where nearly 100 per cent success has been obtained except in the case of some three or four species of bacteria that are especially difficult to stain.     

Notes and queries

A method for making permanent whole mounts of flat and round worms is described. The specimens are mounted in a drop of acid fuchsin lacto-phenol on a slide and warmed for 6 hours at 60°C. The acid fuchsin is replaced by light cotton-blue (anilin blue, W. S.) in lacto-phenol, till the desired contrast is obtained. After this, the forms are mounted in pure lacto-phenol, using the coverglass. The margin of the coverglass is sealed with the sealing media devised by Dade and Waller (equal parts of damar balsam and beeswax)     

Whole Mounts of Flat and Round worms for Morphological Studies

A method for making permanent whole mounts of flat and round worms is described. The specimens are mounted in a drop of acid fuchsin lacto-phenol on a slide and warmed for 6 hours at 60°C. The acid fuchsin is replaced by light cotton-blue (anilin blue, W. S.) in lacto-phenol, till the desired contrast is obtained. After this, the forms are mounted in pure lacto-phenol, using the coverglass. The margin of the coverglass is sealed with the sealing media devised by Dade and Waller (equal parts of damar balsam and beeswax)     

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.     

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.     

An Evaluation of the Metachromasy of Anionic Dyes. I. Visual Observations on Tissue Sections

Thirteen dyes of the azo (benzopurpurin, Congo red, trypan blue, chromotrope 2R, orange G), indigoid (indigocarmine), triphenylmethane (acid fuchsin, aniline blue, light green, methyl blue), and xanthene (eosin B, eosin Y, erythrosin B) groups were applied under standard conditions to a variety of human, rabbit, rat, mouse and frog tissues in paraffin sections. Sections were examined for color changes which might indicate metachromatic reactions analogous to the metachromasy of cationic dyes. Disazo and xanthene dyes showed shifts in hue, with some qualification on the shifts of xanthenes. Metachromatic shifts of anionic dyes were generally of low order compared to those of cationic dyes. Nuclei, erythrocytes, inner elastic laminae of arteries, keratinous structures, and certain areas in the ground substance of connective tissue most often elicited metachromasy. It is suggested that basic proteins are responsible for the metachromatic reactions. Equally interesting areas were those staining poorly (cartilage matrix, most types of mucus), since these are sites of highly acidic substances capable of binding proteins.