A Cresyl Fast Violet Stain for Bacteria and Fungi in Tissue

The cresyl fast violet staining method was modified to eliminate differentiation. Paraffin sections from tissues fixed in Zenker-formol were stained in a 1% aqueous solution of cresyl fast violet (Chroma), adjusted to pH 3.7 with acetic acid, washed in running tap water, dehydrated and covered. Because basophilia increases with time of fixation or storage in formalin or Kaiserling's fluid, dilution of the dye solution to 0.5-0.1% is recommended for such material. Bacteria, nuclei, Nissl substance, and lipofuscin were colored dark blue; fungi, blue to purple; and cytoplasm and muscle fibers, light blue. Collagen and reticulum fibers were only faintly stained. Thus, microorganisms were easily visible against the lightly colored background. In formalin-fixed material, bile pigment was colored olive green. Because this method does not require differentiation, it gave uniform results even in the hands of different users. Little or no fading was observed in sections stored for more than 2 yr.     

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.     

THE EFFECT OF ACETATE BUFFER MIXTURES,ACETIC ACID,AND SODIUM ACETATE,ON THE PROTOPLASM,AS INFLUENCING THE RATE OF PENETRATION OF CRESYL BLUE INTO THE VACUOLE OF NITELLA

When living cells of Nitella are exposed to a solution of sodium acetate and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, a decrease in the rate of penetration of dye is found, without any change in the pH value of the sap. It is assumed that this inhibiting effect is caused by the action of sodium on the protoplasm. This effect is not manifest if the dye solution is made up with phosphate buffer mixture at pH 7.85. It is assumed that this is due to the presence of a greater concentration of base cations in the phosphate buffer mixture. In the case of cells previously exposed to solutions of acetic acid the rate of penetration of dye decreases with the lowering of the pH value of the sap. This inhibiting effect is assumed to be due chiefly to the action of acetic acid on the protoplasm, provided the pH value of the external acetic acid is not so low as to involve an inhibiting effect on the protoplasm by hydrogen ions as well. It is assumed that the acetic acid either has a specific effect on the protoplasm or enters as undissociated molecules and by subsequent dissociation lowers the pH value of the protoplasm. With acetate buffer mixture the inhibiting effect is due to the action of sodium and acetic acid on the protoplasm. The inhibiting effect of acetic acid and acetate buffer mixture is manifested whether the dye solution is made up with borate or phosphate buffer mixture at pH 7.85. It is assumed that acetic acid in the vacuole serves as a reservoir so that during the experiment the inhibiting effect still persists.     

Dichromatism of bromphenol blue, with an improvement in the mercuric bromphenol blue technic for protein.

Staining of protein in sections using the mercuric bromphenol blue technic is improved by staining with 1% HgCl2 and 0.05% bromphenol blue in 2% aqueous acetic acid for 15 min at room temperature. Rinse slides 20 min in 2 changes of 0.5% aqueous acetic acid. Blot and give 2 fast changes in absolute ethanol with agitation before transferring to xylene. Transfer slide to 0.5% n-butylamine in xylene for a few seconds until the section is blue, then, after 2 changes of xylene, mount in DPX. Spectrophotometric analysis of this blue dye at different concentrations and with or without heparin showed that the reddish hues are due to dichromatism and not metachromasia.     

Dichromatism of Bromphenol Blue, with an Improvement in the Mercuric Bromphenol Blue Technic for Protein

Staining of protein in sections using the mercuric bromphenol blue technic is improved by staining with 1% HgCl₂ and 0.05% bromphenol blue in 2% aqueoua acetic acid for 15 min at room temperature. Rinse slides 20 miu in 2 changes of 0.5% aqueous acetic acid. Blot and give 2 fast changes in absolute ethanol with agitation before transferring to xylene. Transfer slide to 0.5% n-butylamine in xylene for a few seconds until the section is blue, then, after 2 changes of xylene, mount in DPX. Spectrophotometric analysis of this blue dye at different concentrations and with or without heparin showed that the reddish hues are due to dichromatism and not metachromasia.     

Advantages of picrate fixation for staining polypeptides in polyacrylamide gels

When acetic acid-urea polyacrylamide gels with or without Triton X-100 were immersed in 0.1 M Na picrate, pH 7, to which 1/4 vol Coomassie blue staining solution (0.2% in 45% methanol, 10% acetic acid, 45% water) was added, proteins stained rapidly (within a few minutes in gels without Triton and within an hour in gels with Triton) with little or no background staining. Thus protein bands could be observed in a single step with no destaining. The picrate-Coomassie blue method fixed and stained a small peptide (bradykinin, nine amino acids) that was not observed in gels stained with fast green, silver, or Coomassie blue following fixation in 50% trichloroacetic acid. The picrate-Coomassie blue method gave high-contrast bands suitable for densitometry. Gels containing sodium dodecyl sulfate were also stained by the picrate-Coomassie blue method if they were first washed briefly (1 h) in 45% methanol, 10% acetic acid, 45% water, presumably to remove the detergent. These gels also stained rapidly with almost no background.     

Acid Versus Neutral Formalin Solution as a Neurological Fixative

The fixing action of 10% formalin solution alone and with formic, acetic, propionic, butyric, lactic, monochloracetic, dichloracetic, or trichloracetic acid was studied by means of stains with silver, osmic acid and cresyl violet. The following conclusions were reached:

1. In general, better fixation and staining was obtained with acid than without.

2. Less difference was seen in comparing one acid with another than was expected before the experiments were made.

3. Propionic, butyric, and dichloracetic showed no promise of having practical value.

4. Formic and monochloracetic acids as modifiers gave superior stains with osmic acid, while silver and cresyl violet stains of the same material were about equal to those made from formalin-acetic fixed material.

5. Lactic acid caused somewhat more distortion of tissue elements than the others but was compatible with good staining.

6. Acetic acid was most effective in concentrations of 3 to 5% while the stronger acids such as formic, monochloracetic, lactic and trichloracetic were effective in concentrations of 0.5 to 1%.     

Erythrocytes and age. Disintegration of erythrocytes by brilliant cresyl blue in correlation to age

The author describes changes in the disintegration of erythrocytes by brilliant cresyl blue in correlation to age, in rats aged 21, 42, 90-105, 340-360 and 690-720 days. The erythrocytes were incubated for 4 hours in an isotonic NaCl solution, in Krebs-Ringer solution and in each of these solutions plus brilliant cresyl blue. Disintegration in plain NaCl solution was found to be the greatest in the case of erythrocytes from 690- to 720-day-old rats. In the same solution plus brilliant cresyl blue, the rate of disintegration was very high in 21-day-old, 42-day-old and 690- to 720-day-old animals; at 90-105 days it was lower and at 340-360 days it was the lowest. Disintegration of erythrocytes in plain Krebs-Ringer solution was the lowest at 21 and 42 days; in the other age groups it was slightly higher. On adding brilliant cresyl blue, the rate of disintegration rose significantly in 21-, 42- and 690- to 720-day-old animals; at 90-105 days and 340-360 days it was no different from disintegration in plain Krebs-Ringer solution. It can be seen from the results that the rate of brilliant cresyl blue-induced erythrocyte disintegration is dependent on the age of the animals from which the erythrocytes are taken.     

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.     

Bromphenol Blue as A Stain for Muscle Striations

Sections from formalin-fixed muscle are stained 4-24 hr with a 0.05% solution of bromphenol blue in 2% acetic acid, rinsed with water and placed in 0.5% acetic acid for 5-10 min. They are then treated 30 sec with tap water substitute (KHCO₃, 0.2 gm; MgSO₄, 2 gm; distilled water, 100 ml), rinsed, dehydrated in alcohol, cleared in xylene and covered in a polystyrene mountant Striatums of both cardiac and skeletal muscle fibers are fully resolved under oil immersion, against the blue background of the other parts of the fibers.     

Nile Blue Histochemical Method for Phospholipids

The technic recommended is: Fix 6-12 hr. in 10% formalin containing 1% CaCl₂. Cut frozen sections without embedding or after gelatin or carbowax. Stain 90 min. at 60°C. in saturated aqueous Nile blue sulfate, 500 ml. plus 50 ml. of 0.5% H₂SO₄, boiled 2 hr. before use. Rinse in distilled water, and place in acetone heated to 50°C. Remove the acetone from the source of heat and allow the sections to remain 30 min. Differentiate in 5% acetic acid 30 min., rinse in distilled water, and refine the differentiation in 0.5% HCl for 3 min. Wash in several changes of distilled water and mount in glycerol jelly. Results: phospholipids - blue; everything else - unstained. Counterstaining nuclei with safranin is optional, but if done, it preferably precedes the Nile blue and is then differentiated by the acetic acid. The histochemical principles on which the method is based are as follows: (1) The calcium compounds of phospholipids combine with the oxazine form of Nile blue sulfate and survive subsequent treatment; (2) neutral lipids are dissolved out by acetone; (3) proteins and other interfering substances are destained by the acetic acid and hydrochloric acid baths.     

A Simplified Stain for Hemoglobin in Tissues Or Smears Using Patent Blue

The following technic, based on the patent blue V hemoglobin reaction, is useful for identifying hemoglobin in tissue fixed in neutral formaldehyde solution and embedded in paraffin:

Stain the deparaffinized, hydrated sections 3 to 5 minutes in the working reagent, prepared by adding 2 ml. of glacial acetic acid and 1 ml. of 3% hydrogen peroxide to 10 ml. of the filtered stock solution (1 g. patent blue, 10 g. zinc powder, and 2 ml. glacial acetic acid). Counterstain 30 to 60 seconds in 1:1000 safranin solution in 1% acetic acid, rinse, dehydrate with alcohols, clear in xylene and mount in clarite. Total time required, 37 minutes.

Blood and tissue and smears may be stained, following fixation in methyl alcohol, by applying the working reagent as above.     

Photooxidation of methionine with immobilized methylene blue photooxidizer

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37°C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4°C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94% while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.     

Additional Schiff-Type Reagents for Use in Cytochemistry

Twenty-four new Schiff-type reagents were discovered in a survey of 140 different dyes. These dyes include acid fuchsin, acridine yellow, acriflavine hydrochloride, azure C., Bismarck brown R, Bismarck brown Y, celestine blue B, chrysoidine 3R, chrysoidine Y extra, cresyl violet, crystal violet, gentian violet, methylene blue, neutral violet, phenosafranin, phosphine GN, proflavine, toluidine blue O, and toluylene blue. Positive results obtained with crystal violet and a few samples of methylene blue are considered due to impurities. Various chemical extractions, aldehyde blocking reagents, and enzymatic treatments were used to verify the aldehyde specificity of the above dye-SO₂, reagents as well as azure A, brilliant cresyl blue, neutral red, safranin O, and thionin which have been mentioned by other workers. These reagents were tested in the Feulgen reaction for DNA and the PAS reaction for polysaccharides. Absorption curves were obtained from individual nuclei stained for DNA. The absorption peaks ranged from 450 mμ, to 630 mμ. depending on the dye studied. The Feulgen reaction could be followed by the PAS reaction or vice versa in mouse intestine using reactive dyes of complementary colors. The evidence indicates that a potential Schiff-type reagent must have at least one free NH₂ group on the dye molecule.     

Photooxidation of methionine with immobilized methylene blue as photooxidizer.

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37 degrees C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4 degrees C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94%, while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.     

A Combined Hcl-Acetic-Alcohol Fixation and Hydrolysis Followed by Cresyl Violet Staining for Mosquito Chromosome Spreads

Mosquito tissues of cytogenetical importance were dissected out on a slide in 0.65% NaCl, under a dissecting microscope, and treated about 30 sec in a drop of 1:3 Carnoy's fixative diluted 1:19 with distilled water. Fixing and hydrolysis was done by a single step in a mixture consisting of: glacial acetic acid, 1; ethanol 96%, 3; HCl conc., 2; and distilled water, 2 (v/v) for 2-6 min at 20-25 C. The specimen was then rinsed with the acetic-alcohol fixative and covered in a drop of 1% cresyl violet in 50% acetic acid under a coverslip coated with Mayer's albumen. Washing was performed immediately by adding water dropwise to one side of the coverslip and drawing the fluid from the other side with absorbent paper. The preparation could be used either as a temporary slide or made into a durable mount. The DNA-containing bands of the giant polytenic chromosomes stained dark violet; interband regions, weakly stained or colourless against a clear background. Mitotic and meiotic figures in gonadal cells stained selectively dark violet or violet with a practically unstained cytoplasm.     

A differential staining technique for simultaneous visualization of mitotic spindle and chromosomes in mammalian cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanol:acetic acid solution containing 4 mM MgCl2 and 1.5 mM CaCl2 at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

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.     

Culture and Slide Preparation of Leukocytes from Blood of IMACACA

Leukocyte-rich plasma is recovered from monkey blood by centrifugation. Portions of the plasma are planted in a medium consisting of 70% NCTC 109, 10% F 10, 15% fetal bovine serum, 5% human cord serum, and phytohemagglutinin (0.1 ml/5.0 ml of medium). Incubation is 48-72 hr, followed by colchicine 24 hr, hypotonic treatment with water 1-1 1/4 hr, and fixation in absolute methanol-glacial acetic acid (3:1). Permanent spreads are made by ejecting drops of suspension onto iced slides and flame drying. Staining is done in cresyl violet acetate. Because of some inconsistencies encountered in the spreading of monkey cells, alternate methods have been suggested for trial until one is found that works uniformly for the technician.     

Acid alcoholic brilliant cresyl blue stain for gastric and other mucins

Summary Some but not all samples of brilliant cresyl blue (6-methyl-7-dimethylamino-2-phenoxazin chloride) under C. I. No. 51010 in Conn's Biological Stains when dissolved at 1% level in 50–70% alcohol containing 1% concentrated (12 N) hydrochloric acid, stain (in 30 min) a wide variety of human and laboratory animal mucins blue black on an almost unstained background. The mucoprotein of the gastric surface epithelium and of the peptic gland neck cells of several species reacts strongly. A 16 hr 60° C methylation in 0.1 M methyl-sulfuric acid in methanol is required to block the staining of these gastric and some intestinal mucins, while 1–2 hr intervals suffice to prevent the staining of mast cells, cartilage and metachromatic sulfomucins generally. Saponification (1% KOH/70% alcohol, 20min) does not restore staining in either location group, indicating that sulfate mucins are probably reacting in both.Most other basic dyes fail to stain mucins from acid alcohol solutions: azure A, toluidine blue, resorcin blue, orcein, resorufin, azoresorufin brown, azolitmin, lacmoid, gallocyanin, Nile blue, methylene green, pararosanilin, crystal violet, Victoria blue R. Some staining occurred with one of three lots of Victoria blue B, with two lots of Victoria blue 4 R and with one lot each of Bernthsen's methylene violet, elastin violet PR and elastin purple PP.The stain may be preceded by the Feulgen reaction to give red nuclei, or followed by a brief collagen stain in an alcoholic acid fuchsin (0.05–0.1%), picric acid (1.5%) solution.Presented before the Symposium of the Histochemische Gesellschaft in Hamburg, 28. September 1968.Supported by National Cancer Institute Grant No. C-4816, National Institutes of Health.