Staining with Phloxine

A double stain with Magdala red and anilin blue has sometimes given very satisfactory results; but, just as often, has been entirely worthless. The reason for the discrepancy seems to be that stains sold under the name of Magdala red are of various composition, some of them containing no Magdala red at all. The standardized stain phloxine seems to be identical with successful lots of Magdala red and results are rather uniformly successful. Detailed directions for staining with phloxine and anilin blue will be published in a forthcoming number of Stain Technology.     

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.     

Staining Bacteria and Yeasts with Acid Dyes

Various acid dyes prove satisfactory for the routine staining of bacteria. Those used are acid fuchsin, anilin blue w. s., fast acid blue R, fast green FCF, light green, orseilline BB, erythrosin, phloxine and rose bengal. Acid fuchsin, fast green, anilin blue, and orseilline are especially recommended. Phenolic solutions of the dyes, acidified with acetic acid, with the addition of ferric chloride to those containing acid fuchsin, anilin blue, fast green or light green, are used. Procedures are given in detail for staining or demonstrating vegetative cells, resting and germinating spores, capsules, sheaths and glycogen in bacteria; germinating and conjugating spores of yeast; and for counterstaining after acid fast or Gram staining. The principal advantages of using acid dyes are better differentiation, and less tendency for slime amd debris to take the dye.     

Haematoxylin-Phloxine-Alcian Blue-Orange G Differential Staining of Prekeratin, Keratin and Mucin

This is a modification of Kreyberg's stain with Alcian blue 8GS used to stain acid much while phloxine B and orange G stain keratin and prekeratin. Procedure: Dewax formalin-fixed paraffin sections in xylene and hydrate through alcohol. Stain in Mayer's haemalum, 10 min; blue in tap water; wash in distilled water; stain in 1% phloxine, 3 min; wash in running water, 1 min; wash in distilled water; stain in 0.5% aqueous Alcian blue in 0.5 acetic acid, 5 min; wash in distilled water; stain in 0.5% orange G dissolved in 2.0% phosphotungstic acid, 13 min; dehydrate quickly in 2 changes of 95% alcohol and 2 changes of absolute alcohol; clear in several changes of xylene; mount in a synthetic resin. Acid mucopolysaccharides are stained turquois blue; prekeratin and keratin are orange to red orange.     

Plant-Virus Differentiation by Trypan-Blue Reactions within Infected Tissue

Trypan blue has proved effective for demonstrating the presence of certain plant viruses within infected tissues. The amorphous and crystalline inclusions which constitute cytological evidence of viruses stain proportionately. The effects produced by different viruses react differently to the stain and those inclusions which do not absorb trypan blue tend to stain with phloxine. This selective staining is the basis for using trypan blue singly and in combination with phloxine as standardized procedures for demonstrating and differentiating cytological evidence of plant viruses. These tests are very rapid and are especially applicable to temporary mounts of living tissue but permanent mounts can be made from material fixed in formalin.     

A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

Phloxine-Methylene Blue Staining of Formalin-Fixed Tissue

It is at present difficult to obtain a good phloxine-metbylene blue stain on formalin-fixed tissue. When phloxine has been used, it is washed out in the process of staining with methylene blue and differentiating with colophony (rosin). In the original technic of Mallory, Zenker's fixation is used. The tissue is first stained with a 2.5% aqueous solution of phloxine, then with a solution of 1% methylene blue plus 1% azure II and differentiated in colophony.¹     

Suggested Counterstains for Davenport Reduced Silver Preparations of Peripheral Nerves

—Peripheral nerves which have been fixed in a mixture of formaldehyde and acetic acid and stained according to the method of Davenport can be successfully counterstained for demonstration of myelin sheaths and stroma. After mounted sections have been silvered, reduced and toned, the coating of nitrocellulose is removed by passing thru two changes of acetone. Following brief washes in 100,95,85 and 75% alcohols they are stained in an acidified aqueous solution of azo carmine for 30 to 60 minutes. Excess azo carmine is extracted with anilin alcohol followed by acetic alcohol after which the sections are mordanted for 15 to 60 minutes in a 5% aqueous solution of phosphotungstic acid. Without washing they are transferred to a stain mixture of either anilin blue and orange G (acidified) or light green and orange G (acidified) where they remain from 1 to 5 hours. After destaining in 95% alcohol and dehydration in absolute alcohol the sections are mounted in dammar. Result: axons stain black; sheath and fibroblast nuclei, red; myelin sheaths, orange; and connective tissue, blue or green. When the counterstains are applied to ganglia, cytological details of individual cells are demonstrated.     

Red Food Coloring Stain: New,Safer Procedures for Staining Nematodes in Roots and Egg Masses on Root Surfaces

Acid fuchsin and phloxine B are commonly used to stain plant-parasitic nematodes in roots and egg masses on root surfaces, respectively. Both stains can be harmful to both the user and the environment and require costly waste disposal procedures. We developed safer methods to replace both stains using McCormick Schilling red food color. Eggs, juveniles, and adults of Meloidogyne incognita stained in roots with red food color were equally as visible as those stained with acid fuchsin. Egg masses stained with red food color appeared as bright-red spheres on the root surfaces and were highly visible even without magnification. Replacement of acid fuchsin and phloxine B with red food color for staining nematodes is safer for the user and the environment, and eliminates costly waste disposal of used stain solutions.     

Displacement.

Displacement is a noncommital term for the reactions that occur when slides previously stained in phloxine or rose Bengal are immersed for varying lengths of time in a solution of another dye in ethyl Cellosolve. In most histotechnic texts Lendrum's (1947) phloxine-tartrazine is given as the stain for acidophilic inclusion bodies. However the lack between the phloxine and tartrazine has been a serious limitation. A number of dyes were tried as possible substitutes for the tartrazine. A rose Bengal-Bismark brown Y procedure was developed which stains similarly to Lendrum's phloxine-tartrazine and which does have the needed contrast. After staining for 10 min in 1% aqueous rose Bengal and rinsing in isopropyl alcohol slides are placed for 20, 30, 40 and 50 min in 0.05% Bismark brown Y in ethyl Cellosolve. In various tissues and structures the rose Bengal is sequentially displaced by the Bismark brown Y. Thus collagen loses the red stain after 30 min while acedophilic structures like sperm heads and Paneth cell granules retain the red stain after 50 min in the displacement solution. The results are strikingly similar to staining with alkaline Biebrich scarlet.     

Displacement

Displacement is a noncommital term for the reactions that occur when slides previously stained in phloxine or rose Bengal are immersed for varying lengths of time in a solution of another dye in ethyl Cellosolve. In most histotechnic tests Lendrum's (1947) phloxine-tartrazine is given as the stain for acidophilic inclusion bodies. However the lack of contrast between the phloxine and tartrazine has been a serious limitation. A number of dyes were tried as possible substitutes for the tartrazine. A rose Bengal-Bismark brown Y procedure was developed which stains similarly to Lendrum's phloxine-tartrazine and which doer have the needed contrast. After staining for 10 min in 1% aqueous rose Bengal and rinsing in isopropyl alcohol slides are placed for 20, 30, 40 and 50 min in 0.05% Bismark brown Y in ethyl Cellosolve. In various tissues and structures the rose Bengal is sequentially displaced by the Bismark brown Y. Thus collagen loses the red stain after 30 min while acidophilic structures like sperm heads and Paneth cell granules retain the red stain after 50 min in the displacement solution. The results are strikingly similar to staining with alkaline Biebrich scarlet.     

A Rapid Phloxine-Methylene Blue Oversight Stain for Formalin-Fixed Material

A rapid method is described, which yields brilliant and selective differential staining with phloxine-methylene blue. Aqueous phloxine (1%) is treated with hydrochloric acid (1 ml/l gm of phloxine) and the water-insoluble precipitate is washed, dried, and dissolved in acidified 95% ethyl alcohol. Slides are stained for several minutes in this solution (0.2%) followed by brief staining in the usual azure-methylene blue solution and differentiated with colophonium-alcohol. The method eliminates the necessity for prolonged staining at elevated temperatures, reducing the total staining procedure to approximately 15 min. In addition, controlled differentiation with colophonium-alcohol can be carried out on formalin-fixed material without loss of phloxine. The selectivity and tinctorial attributes of phloxine are also considerably improved.     

Mallory's Connective Tissue Stain Following Hematoxylin

The following combination of hematoxylin with Mallory's connective tissue stain is useful in bringing out nuclei as well as in differentiating tissue:

Slightly overstain in Mayer's hematoxylin (50 g. potassium alum and 0.2 g. sodium iodate added to 1 liter 0.1% aqueous hematoxylin). Wash; and stain 30 seconds to 1 minute in 0.04% aqueous acid fuchsin-Stain 4 minutes in: 0.5 g. anilin blue and 2 g. orange G dissolved hi 100 cc. of 1% aqueous phosphomolybdic acid. Pass thru 95% alcohol to absolute; clear in xylol and mount in balsam.     

Negative Stains in the Demonstration of Bacteria

Since certain objectionable features have been observed in the old technics of preparing negatively stained bacteria slides, modifications of these methods have been attempted, and studies have been made of various dyes not heretofore described as negative stains. India ink, nigrosin, indulin, Congo red, Poirier's blue, anilin blue (methyl blue), China blue, blue de Lyon, blue de Lyon O, and night blue are recommended. Dyes of the same name, but sold by different companies, often present different effects.     

Phloxine As An Histologic Stain, Especially In Combination With Hematoxylin

A staining schedule employing phloxine as a counter-stain to Erlich's acid hematoxylin is presented. Fixation is best with Zenker's fluid, although formalin can be used. The technic is similar to the standard hematoxylin-eosin formulae but because of the staining advantages of phloxine over eosin, the technic is simpler, and quicker, resulting in clearly differentiated sections which do not fade as soon as do eosin-stained slides. A brief summary of the uses of phloxine as a biological stain is given and its advantages over eosin are discussed.     

The Recognition of Lipolytic Activity with the Microscope

In view of its probable wide applicability, it seems desirable to publish a note on a simple technic for the recognition with the microscope of the action of lipase. In brief, the method is to make an emulsion of neutral fat previously stained with a red Sudan stain, subject some of the emulsion to the action of the supposed lipase for an appropriate time and then examine with the microscope a recovered drop of the emulsion in a solution of Nile blue sulfate. It has long been known that Nile blue sulfate stains liquid neutral fats a reddish color and fatty acids blue.     

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.     

Luxol Fast Blue Mbs and Phloxine; a Stain for Mitochondria

The applicability of Luxol fast blue MBS as a 0.1% solution in 0.05% acetic acid to the staining of mitochondria, first recognized in rat kidney by Shanklin and Nassar (Stain Techn., 34: 257-60. 1959), was confirmed in various organs (formalin-Zenker and Regaud's fixations; paraffin embedding) of the mouse and bullfrog. In liver cells and in the epithelium of renal tubules, mitochondria were stained green, selectively and clearly. The dark cells of the renal tubules and the middle piece of sperms in both animals were conspicuously demonstrated by their dense assemblages of green granules. The periodic acid-Schiff procedure proposed by Shanklin and Nassar as a counterstain was replaced by staining in 0.5% aqueous phloxine, 2-3 min; differentiation in 5% phosphotungstic acid, 2 min; and washing in water, 5 min. This simplified and accelerated the techique, and gave a better color contrast. Advantages of Luxol fast blue MBS and phloxine staining over traditional methods for mitochondria in paraffin sections are: durability of the stain, high specificity, simplicity of procedure, and constant result.     

A simple polychrome stain for conventionally fixed Epon-embedded tissues.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

An Easily Controlled Regressive Trichromic Staining Method

Three modifications of Mallory's connective tissue stain are described and some features of the action of picric acid are discussed.

In the first and most critical method the nuclei are stained in an iron hematoxylin and then differentiated in a picric acid solution containing orange G. This not only differentiates the nuclei, but stains all other elements yellow. The section is then washed in running water to remove the yellow color from all tissues except those which are to remain yellow in the final preparation (usually the erythrocytes). The section is next stained in an acid fuchsin mixture and then differentiated until the desired depth and contrast is obtained. Staining in anilin blue follows and this in turn is differentiated to suit. The section is then dehydrated and mounted.

In the second method the nuclei are stained in hemalum (e.g. Harris's) for a short time; the section is then rinsed and immersed in a mixture of picric acid and acid fuchsin and thereafter is differentiated; it is next passed into anilin blue w. s. and then differentiated and mounted as before. This is less critical than method I, but can be applied to large batches of slides at a time.

The third method is a one-solution method. After staining the nuclei in hemalum, the section is immersed in the “Picro-Mallory” solution, differentiated briefly, dehydrated and mounted. This modification, while being the least critical, is most suitable for routine use when the tissues have been fixed in a fluid containing chromate; the other commonly used fixatives, while giving useful results, are not so good.