The Combined Gram-Pappenheim Stain as Modified for Films and Paraffin Sections

A combination of the Gram-Pappenheim stains for the examination of gonorrheal pus, cellular exudate and paraffin sections of formalin-fixed tissues has been described elsewhere (Scudder and Lisa, 1931). The crystal violet solution was made stable for the first time by employing phosphate buffers on the acid side of neutrality, and a stable counterstain was prepared for the first time from National Aniline dyes, ethylated methyl green and pyronin yellowish. Original findings were demonstrated by means of color plate I and II (Scudder, 1931) to show gonococci, pneumococci and cells in smears, and formalin-fixed tissue brought down to water in the usual way. A new color plate is published herewith to show the microscopic appearance of cells, Gram-positive and Gram-negative bacteria, higher bacteria, fungi and spermatozoa in the study of genitourinary and gynecological cases. The method has a value in the field of medical jurisprudence. Crystals were well demonstrated, especially those resulting from sulfa drug therapy. The National Aniline methyl green batches numbered NG 10, 11, 13 to 19, and their batches of pyronin numbered NP 5 to 10 were found consistently stable. Earlier dyes were found either too purple or too blue for the technic and the most satisfactory dyes were found to require a ripening time of several days and could be prepared in amounts of from 1 to 4 liters and stored indefinitely without preservatives.     

Schiff-Type Reagents in Cytochemistry. 2. Detection of Primary Amine Dye Impurities In Pyronin B and Pyronin Y(G)

Many batches of pyronin B (C.I. 45010), pyronin Y or G (C.I. 45005), and acridine red (C.I. 45000) produce positive Feulgen or PAS reactions when their 0.25% solutions are saturated with SO₂ and used on acid-hydrolyzed or periodate-oxidized tissue sections. These dyes behave as Schiff-type reagents and stain aldehyde-containing structures orange, brown, pink, red, or violet, depending on the particular batch used. The most frequent contaminants are violet and are nonfluorescent. Aldehyde groups are stained by these dyeSO₂ solutions as is shown by using unhydrolyzed controls in the Feulgen reaction and unoxidized controls in the PAS reaction, and by dye solutions lacking SO₂. Other procedures included reactions with aldehyde-blocking reagents, treatment with deoxyribonuclease and diastase, and extraction of nudeic acids with trichloroaeetic acid. The standard Schiff reagent was used in the Same procedures as a basis for comparing results. Since the Schiff-aldehyde reaction requires a dye with a primary amine group and since true pronins contain only secondary or tertiary amines, the positive histochemical results are evidently caused by dye contaminants possessing primary amine groups. The PAS reaction is more sensitive than the Feulgen reaction in detecting dye contaminants. Tissues used were chiefly formalin-fixed mouse intestine and ascites cells. Seventy-five commercial pyronins were studied from 21 different firms. Among 19 batches of pyronin B, 14 were found to contain primary amine dye contaminants. Among 39 batches of pyronin Y(G), 19 contained similar primary amine dye contaminants. Of the 8 batches of acridine red tested, 7 were found to contain primary amine dye contaminants. Nine commercial mixtures of methyl green-pyronin were studied and 4 were found to be likewise contaminated, but these reactive dye contaminants in them are apparently not associated with methyl green. A tabulated summary of the pyronin batches containing primary amine contaminants, and a list of sources and distributors of pyronin dyes are included.     

A Differential Stain Favorable to the Diagnosis of Neisserian Infection

A differential Gram stain has been evolved which incorporates the combined features of the original Gram and Pappenheim methods. National Aniline crystal violet and new methyl green and pyronin are the dyes preferred. The iodine mixture of Kopeloff and Beerman is a satisfactory mordant and Merck's pure technical acetone is an excellent differentiating agent. A system is established by means of the dyes and reagents which form a physicochemical equilibrium, provided pure dyes are employed, and the technic is carried out with precision. Gram-positive bacteria are coated by means of buffered crystal violet solution and the iodine-sodium hydroxide solution precipitates the crystal violet from other substances. The dye-iodine precipitate is readily dissolved by pure acetone. Iodine green, a pure derivative of crystal violet has the effect of noninterference in the technic and has selective action upon nuclear substance. Pyronin has affinity for Neisserian organisms primarily and acts as an inert substance upon most other proteins, (except cytoplasm of eosinophils, lymphocytes, plasma cells, and endothelial cells). The following technic is recommended:

Stain air-dry films 3 to 5 minutes in a 1% solution of crystal violet in 10 parts of Clark and Lubs' phosphate buffer of pH 6.6 to 7.0 and 90 parts water. Decant and flush with 2% iodine in N/10 NaOH. Decant and decolorize in acetone 10 seconds or less. Air dry and counterstain 1 1/2 to 2 minutes with methyl-green-pyronin (2 parts 2% aqueous methyl green National with one part 0.3% aqueous pyronin yellowish). Wash and air dry. Oil of Bergamot is preferable to xylene as a clearing agent. Best results are obtained if each slide is handled separately as for staining blood films.     

A new rapid method for selective extraction of RNA from fixed mammalian tissues.

Treatment of formalin-fixed mammalian tissues with concentrated or 50% phosphoric acid at 5 degrees C for 20 and 50 min. respectively reveals complete extraction of RNA as judged by methyl green followed by staining with pyronin. This procedure also causes depolymerisation of DNA as indicated by the red staining of the nuclei. Sections treated with concentrated phosphoric acid at 5 degrees C for 30 min. causes disruption of the double helical structure of DNA what results in the depression of the pyronin staining. Similarly treated sections show Feulgen positive nuclei. Treatment of sections in 25 % phosphoric acid at 60 degrees C for 15 min. followed by staining with methyl green and pyronin show red nuclei, nucleoli and the cytoplasm. This indicates that extraction of RNA is only possible in cold and not at elevated temperature.     

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time,dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.     

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time, dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.     

Staining Aspirated Human Bone Marrow with Domestic Wright Stain

The method employs the domestic Wright stain for the staining of aspirated human bone marrow. Freshly distilled water, pH 6.0 to 6.4, is used. Wright stain, 0.5 cc, is placed upon the air-dried preparation and permitted to act for two minutes. The stain is then diluted with 2 cc. distilled water and permitted to act for from 5 to 10 minutes. After washing off the stain with distilled water, the preparation is placed into a decolorizer (acetone 0.5 cc, pure methyl alcohol 5.0 cc, and 100 cc. distilled water, pH 6.0 to 6.4) for differentiation from 1 to 5 seconds, rinsed, washed under running water and permitted to air-dry. A well stained and differentiated preparation shows the “Romanovsky effect”, and the sharpness of minute structures obtained compares favorably with control preparations stained with German dyes.

The bone marrow should be prepared as described. The Wright stain marketed by the National Aniline and Chemical Co., N. Y. was found to be reliable as regards staining quality of registered batches. One photomicrograph, showing bone marrow cells from pernicious anemia, is included.     

Spectrophotometry of Dyes: 1. Methyl Green. 2. Pyronin

Comparisons of absorption peaks of seven samples of methyl green showed that two different types of the dye were represented. One type (2 samples) had the visible peak near 617 mμ; the other (4 samples) near 630 mμ, while one sample was intermediate in spectral characteristics. Using these findings as a means of differentiating between heptamethyl and hexamethylethyl pararosanil-in is suggested. The Y and B forms of pyronin were found to be readily distinguishable by comparing their absorption maxima (Y, 546 mμ, B, 557-8 mμ). A check on the application of Beer's law of dilution showed that it held (1-3 mg./liter) for pyronin and that the relative effect of dilution was a slow increase with pyronin but a rapid decrease with methyl green.     

Qualitative Analysis by Thin-Layer Chromatography of Some Common Dyes Used in Biological Staining

Thin-layer chromatography will resolve impurities in commercial dyes, and will do so much faster than paper chromatography. Solvent systems consisting of (a) n-propanol: n-butanol: NH₄OH (conc.): H₂O—4:4:1:1; (b) n-propanol: NH₄OH (conc.): H₂O—8:1:1 on silica gel G plates; and (c) n-propanol: NH₄OH (conc.): H₂O-7:2:1 on Adsorbosil plates were found to be the most effective. Dyes studied were azure A, azure B, azure C, methylene blue, toluidine blue O, thionin, pyronin B, pyronin Y, methyl green, crystal violet amido black 10B and buffalo black (NBR).     

Spectrophotometry of Dyes: 1. Methyl Green. 2. Pyronin

Comparisons of absorption peaks of seven samples of methyl green showed that two different types of the dye were represented. One type (2 samples) had the visible peak near 617 mμ; the other (4 samples) near 630 mμ, while one sample was intermediate in spectral characteristics. Using these findings as a means of differentiating between heptamethyl and hexamethylethyl pararosanil-in is suggested. The Y and B forms of pyronin were found to be readily distinguishable by comparing their absorption maxima (Y, 546 mμ, B, 557-8 mμ). A check on the application of Beer's law of dilution showed that it held (1-3 mg./liter) for pyronin and that the relative effect of dilution was a slow increase with pyronin but a rapid decrease with methyl green.     

Histochemical characterization of the lead intranuclear inclusion bodies

A battery of histological and histochemical techniques was applied on the lead intranuclear bodies that have resuted in the kidneys of adult Wistar male rats receiving lead acetate in their diet to determine their nature. The intranuclear inclusion bodies have stained strongly with xanthene, anthraquinone, and trisulfonated basophilic dyes and weakly with dyes containing both positive and negative radicals, and they have responded negatively to acidophilic cationic dyes. They have also reacted positively to proteins and lead stains, but weakly to lipid stains, and negatively to Feulgen and methyl green pyronin techniques. The intranuclear bodies proved to be lead lipoprotein complexes containing sulfyhydryl groups and are basic in nature with orthochromatic, eosinophilic, argyrophilic, osmophilic, fuchsinophilic, and sudanophilic characteristics.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30-90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30–90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.     

Assessment of Theileria infections in Rhipicephalus appendiculatus ticks collected from the field

Collections of adult Rhipicephalus appendiculatus ticks were made from bait cattle and vegetation at two field sites in areas of Kenya in which East Coast fever caused by Theileria parva is endemic. These ticks, together with two experimentally infected batches of ticks, were examined for infection with Theileria by four methods. Whole salivary glands were stained with methyl green pyronin or Feulgen's stain. Whole ticks were ground in medium, the suspensions were filtered and centrifuged and the treated material was examined microscopically and tested for infectivity by inoculation into cattle. All field collections and experimental batches of ticks were infected with Theileria and all four methods detected the infections. Approximately 1.5% of the ticks in the field collections were found to be infected with Theileria and the treated material from these ticks transmitted T. parva to cattle. It is considered that it will be feasible to survey field infection rates quantitatively by collecting ticks from bait cattle and vegetation for examination by a combination of salivary gland staining and preparation of tick suspensions for microscopy and infectivity tests.     

Comparative evaluation on mouse nasal immunogenicity of arylmethane-, xanthene-, quinone-imine-, and acridine-dye-inactivated Sendai virus vaccines

Twenty-seven kinds of organic dye-inactivated Sendai virus vaccines were prepared by treatment in dark at 23 C for 2 months or more, and selected with the high HA titers as a guide. Their nasal immunogenicities were examined in mice by contact infection and immunofluorescent method, and the relative merits of the dye-inactivants were determined. The strongest protection was elicited with acriflavine-, auramine O-, eosin Y-, neutral red-, night blue-, patent blue V-, thymol blue-, uranin-, and xylene cyanol FF-treated vaccines. Middling protective efficacy was induced by use of erio green B-, malachite green-, methyl green-, proflavine-, pyronin B-, and thionin-inactivated vaccines. Dye-inactivated vaccines that resulted in the weakest protection were Bindschedler's green-, bromothymol blue-, erythrosin B-, ethyl violet-, gallein-, light green SF yellowish-, methyl violet-, new methylene blue N-, phenol red-, rhodamine 6G-, spirit blue- and victoria blue B-treated ones. Serum HI titers developed by nasal vaccination were variable, and rose still more in most vaccinated groups postexposure. Elicitation of the most effective nasal immunogenicity in dye-inactivated vaccines appeared to depend on selective modification of capsid protein or ribose in viral core with dyes possessing definite functions, despite the different molecular structures.     

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.     

Microbiological quality of bottled water sold in retail outlets in Nigeria.

The microbiological quality of four brands of bottled water sold in retail outlets in Nigeria were assessed by routine methods in 90 samples. Samples of two brands were acidic in the pH range 3.5-5.9. Faecal coliforms and streptococci were not recovered from any sample. Heterotrophic plate counts (HPC) numbered 50-800 cfu/ml in two brands, A and B, and 100-87,000 cfu/ml in C and D. Component colony types among the HPC bacteria in brands C and D produced water-soluble, fluorescent pigments on colony count and other agar media, and occurred in 11 of 16 batches: their numbers varied from 60 to 82,000 cfu/ml. Presumptive antibiotic-resistant proportions of the HPC bacteria were also recovered from brands C and D on agar amended with ampicillin, penicillin or streptomycin. Five isolates from the green pigmented colonies, representing five batches of brands C and D were satisfactorily identified as strains of Pseudomonas aeruginosa. These strains resisted between four and nine of 14 antibiotics tested. Resistance to tetracycline was eliminated in one isolate when it was cured by incubation at 42 degrees C for 100 h, but gel electrophoretic resolution of the DNA did not reveal presence of a plasmid. Strains of Bacillus were the second most commonly isolated bacteria; they were the only colony types in most samples with low HPC counts.     

Microbiological quality of bottled water sold in retail outlets in Nigeria

M.T. OGAN. 1992. The microbiological quality of four brands of bottled water sold in retail outlets in Nigeria were assessed by routine methods in 90 samples. Samples of two brands were acidic in the pH range 3.5–5.9. Faecal coliforms and streptococci were not recovered from any sample. Heterotrophic plate counts (HPC) numbered 50–800 cfu/ml in two brands, A and B, and 100–87000 cfu/ml in C and D. Component colony types among the HPC bacteria in brands C and D produced water-soluble, fluorescent pigments on colony count and other agar media, and occurred in 11 of 16 batches: their numbers varied from 60 to 82000 cfu/ml. Presumptive antibiotic-resistant proportions of the HPC bacteria were also recovered from brands C and D on agar amended with ampicillin, penicillin or streptomycin. Five isolates from the green pigmented colonies, representing five batches of brands C and D were satisfactorily identified as strains of Pseudomonas aeruginosa. These strains resisted between four and nine of 14 antibiotics tested. Resistance to tetracycline was eliminated in one isolate when it was cured by incubation at 42C for 100 h, but gel electrophoretic resolution of the DNA did not reveal presence of a plasmid. Strains of Bacillus were the second most commonly isolated bacteria; they were the only colony types in most samples with low HPC counts.     

Detection of Ribonucleic Acid with Pyronin

Pyronin, when used in the methyl green-pyronin stain, is useful in localizing ribonucleic acid (RNA). That it has rarely been used alone is perhaps a result of the observation (Kurnick 1955) that pyronin stains deoxyrobonucleic acid (DNA) of animal tissue when not competitively inhibited by methyl green. The tests described in this note indicate that pyronin alone can be used to demonstrate RNA in fixed plant tissues.     

Chromosomal banding patterns produced by methyl green-pyronin staining after trypsin treatment.

A method is described for producing banding patterns with methyl green-pyronin (MGP) stain in chromosomes of fibrosarcoma cells. 1) The stain was made by mixing equal volumes of 2% aqueous pyronin G, 2% aqueous methyl green, distilled water, and 0.1 M acetate buffer (pH 5.7). 2) Treatment with colcemide and hypotonic KCl (0.075 M) was performed as usual. 3) Metaphase chromosomes were prepared using the flame-drying technique and treated with 0.25% trypsin at 37 C for 45 to 90 seconds. Before staining, the slides were rinsed in PBS, in distilled water, and then were dipped in 0.05 M acetate buffer. 4) Chromosomes were stained for more than 20 minutes, rinsed in distilled water, and hot-air dried. Satisfactory results were obtained in uncontracted metaphase chromosomes. MGP stain has the advantage of permitting much longer trypsin treatment and staining time than the trypsin-Giemsa method while providing satisfactory banding patterns.