Chlorazol Fast Pink B And Trypan Blue Tests for Virus and Other Proteinaceous Inclusions

A 1% solution of chlorazol fast pink B in 0.9% NaCl can be used like trypan blue to detect virus inclusions and proteinaceous entities in peelings from leaves or thin sections taken from living plant tissue. Like trypan blue, a solution of the pink dye causes somatic nuclei to swell and thus facilitates observation of their structure. The two dyes combine into a beautiful differential bicolored stain. Mix 5 ml of 0.5% trypan blue stock solution with 35 ml of 1% chlorazol pink B in 0.9% NaCl. Stain fresh tissue 1-2 minutes. The combination stain is superior to either dye alone for differentiating virus entities.     

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.     

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.     

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.     

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     

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.     

Ultrastructure of trypan blue induced ocular defects: I. Retina and lens

The histological and cytological basis of trypan blue-induced ocular defects were studied using scanning and transmission electron microscopy. Microphthalmic and anophthalmic eyes of 16-day rat fetuses were utilized from dams exposed to a teratogenic dose of trypan blue. Retinal and lenticular anlagen were specifically examined for architectural and cellular changes. Nearly all severely abnormal eyes showed no evidence of retina development: Of 41 such eyes, only two retinal rudiments were observed. Those eyes with mild microphthalmia always demonstrated retinae although architectural changes were present. In every abnormal eye, some degree of lenticular morphogenesis was always present. Lenses were small, displaced in the eye field, and arrested at the lens vesicle stage. Lens cells were markedly undifferentiated and thus lacked most of the cytological features normally present at this developmental stage. Neither retinal nor lenticular rudiments were necrotic despite major architectural and cytological disturbances. The data offer three conclusions: First, the absence of necrosis suggests that trypan blue causes developmental arrest in this eye model; second, absence of retinae is most likely due to primary failure of optic vesicle development; third, lack of lens differentiation is attributed to absence of the retina, the primary lens inducer.     

Intranuclear inclusion bodies within neurons of spinal and cranial ganglia in three cyprinid species

Summary A histological examination of 205 fish representing four cyprinid species from a site 2.5 miles north of Wheeling, West Virginia, on the Ohio River revealed large (2–4 m) cuboidal intranuclear inclusion bodies (NIB's) within neurons in the cranial and spinal ganglia of three species. Because the minnows had been caught during a yearly sampling of fish, an additional 63 minnows were taken the following year. Inclusions were again observed. The NIB's stain strongly with phloxine as well as with Mallory and Giemsa stains, appearing bright red or pink. Various histochemical tests indicated that the inclusions contain protein and lipid but no carbohydrates or nucleic acids. No heavy metals were detected by electron probe analysis. At the ultrastructural level the inclusions exhibit subunits resembling hexagons measuring 326–350 nm. Previously suggested causes for such inclusions include effects of viruses, aging, drugs, cellular transformation, and an altered metabolic state of affected cells.     

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.     

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.     

Trypan Blue as a Nuclear Stain for Plant Material

Although McWhorter¹ and later Rich² mentioned that trypan blue stained the nuclei of plant cells, their procedures were concerned with the demonstration of virus inclusions. The following method was developed and is presented with the emphasis on nuclear staining. The present author hopes that others will try it for comparison with the popular aceto-carmine and aceto-orcein methods.     

A Rapid Stain Method for Detecting Certain Plant Virus Inclusions

The trypan blue method for detecting certain virus crystallin inclusions is greatly facilitated by a pre-stain treatment with a diethyl-ether-ethyl-alcohol solution made up in physiological saline (5-ml. of diethyl ether and 10 ml. of ethyl alcohol diluted to 100 ml. with physiological saline solution). Maceration of the study material in trypan blue solution also gives good results.     

Trypan blue in vivo stains nigral dopaminergic neurons killed by 6-hydroxydopamine

Dopaminergic neurons in the substantia nigra killed by 6-hydroxydopamine were stained in vivo by intracerebral injections of trypan blue. Such staining appeared specific for dead neurons, although a proportion of these retained the ability to stain with Nissl dyes for at least 2 days. Neurons retained trypan blue in vivo for periods of up to 9 days. Trypan blue staining of some neurons outside the substantia nigra demonstrated the use of this dye in determining the degree of non-specific toxicity of 6-hydroxydopamine. Twenty-four hours after infusion of trypan blue almost no background staining was present and individually stained neurons were clearly visible. Thus the use of trypan blue may have a general application as a sensitive method for estimating discrete areas of toxin-induced neuronal death, and for estimating the degree of specificity of a toxin.     

Traditional natural dyeing using crotal

Although McWhorter¹ and later Rich² mentioned that trypan blue stained the nuclei of plant cells, their procedures were concerned with the demonstration of virus inclusions. The following method was developed and is presented with the emphasis on nuclear staining. The present author hopes that others will try it for comparison with the popular aceto-carmine and aceto-orcein methods.     

Trypan blue in vivo stains nigral dopaminergic neurons killed by 6-hydroxydopamine

Summary Dopaminergic neurons in the substantia nigra killed by 6-hydroxydopamine were stained in vivo by intracerebral injections of trypan blue. Such staining appeared specific for dead neurons, although a proportion of these retained the ability to stain with Nissl dyes for at least 2 days. Neurons retained trypan blue in vivo for periods of up to 9 days. Trypan blue staining of some neurons outside the substantia nigra demonstrated the use of this dye in determining the degree of non-specific toxicity of 6-hydroxydopamine. Twenty-four hours after infusion of trypan blue almost no background staining was present and individually stained neurons were clearly visible. Thus the use of trypan blue may have a general application as a sensitive method for estimating discrete areas of toxin-induced neulonal death, and for estimating the degree of specificity of autoxin.     

Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species

A simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of the vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple–dark pink while acrosome-free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37°C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37°C resulted in a decrease over time in the percentage of live acrosome-intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.     

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.     

A triple-stain technique for evaluating normal acrosome reactions of human sperm

A triple-stain technique has been developed to score normal acrosome-reacted human sperm in fixed smears. Live and dead sperm are first differentiated using the vital stain trypan blue. Sperm are then fixed in glutaraldehyde, dried onto slides, and the postacrosomal region and acrosome are differentiated using Bismark brown and Rose Bengal. Slides are examined at 1,000 X with a bright-field microscope and assessed for 1) the percentage of sperm that were alive at the time of fixation and 2) the percentage of sperm that had undergone normal acrosome reactions. Experiments are included that show that trypan blue is a reliable stain for dead sperm and that Rose Bengal stains only sperm having intact acrosomes. This technique may have applications in experimental and clinical studies on sperm capacitation, acrosome reactions, and fertilization in laboratory and domestic animals as well as in man.     

ALKALINE RIBONUCLEASE ACTIVITY INCREASE IN RAT KIDNEY CORTEX AND LIVER AFTER TRYPAN BLUE AND OTHER AZO DYES ADMINISTRATION

Acid azo dyes, most of them naphtholdisulfonic acid derivatives, were given intraperitoneally to rats and their effect on "alkaline" ribonuclease activity was studied in total homogenates of kidney cortex and liver. Acid treatment was used to release bound enzyme activity. Several of the dyes, including trypan blue, increased RNase activity in both organs 3 days after administration of single doses, while others, like Evans blue, were inactive. Activity was apparently bound to the sulfonic substitution in the 3, 6 positions in the naphthalene rings, substitutions in the benzidine rings being not critical. All of the active and most of the inactive compounds were taken up by tubule cells of kidney cortex and by reticular and parenchymal cells of liver. While the effect on both liver and kidney was obtained 1 day after trypan blue administration, RNase remained increased for only about 3 days in the first organ, and for at least a month in the second. However, repeated trypan blue doses increased liver enzyme activity for at least 9 days. Serum RNase activity was decreased after trypan blue administration. Ethionine administration together with trypan blue markedly blocked the effect of the dye on liver RNase activity; simultaneously given methionine partially reversed the action of the antimetabolite. This suggests that de novo synthesis of RNase is induced in liver by trypan blue. The action of ethionine on the kidney RNase response to trypan blue was less marked although significant; in view of the possible kidney uptake of the plasma enzyme, interpretation of this finding must be postponed. Results are discussed with reference to the mechanism of the structural specificity of the compounds used, cytological localization of the dyes and their mechanism of action on liver and kidney RNase.     

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.