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.     

A Flagella Staining Technic for Soil Bacteria

In an attempt to stain the flagella of soil bacteria, many of which have flagella so fine that they are hard to stain by most methods, a technic was developed which combines the best points of Hofer and Wilson's method with that of Bailey as developed by O'Toole. Satisfactory preparations have been obtained for organisms of the genera Pseudomonas, Phytomonas, Alcaligenes, Escherichia, Azotobacter, and Bacillus. This technic, therefore, is recommended as a rapid and constant method for routine flagella staining of all motile aerobic organisms; it combines Hofer and Wilson's method of cleaning the slides with O'Toole's technic of spreading the smears and with a modification of Bailey's mordant.     

A Stable, High-Contrast Mordant for Hematoxylin Staining

Small, quantities of sulfuric acid will stabilize iron mordants, used in hematoxylin staining, by preserving these solutions against oxidation. The presence of acetic acid in the mordant improves the specificity of the stain. A stable, high-contrast mordant is obtained when both acids are combined with ferric-ammonium sulfate. This mordant, used in combination with fresh alkaline solutions of hematoxylin, has been found especially effective in the staining of certain nuclear and cytoplasmic components of plant cells.     

Staining Apparent Nuclear Material in Bacillus Cereus, Neisseria Catarrhalis and Other Cocci

The method of staining involves the principle of reduction and oxidation of some cell component. The reduction is a function of the mordant and the oxidation that of the stain. Three areas of the cells may be differentiated depending upon the condition of the mordant. The wall may remain unstained, while that of the cytoplasm stains a light red and structures within this a deep red. The latter structures only may be stained by proper adjustment of the mordant.     

Some Staining Methods for Bacteria and Yeasts

For staining flagella of bacteria use actively motile organisms 20 to 24 hours old, allow to diffuse in sterile water 20 to 30 minutes, transfer droplets of the suspension to clean slides and let evaporate without spreading. Then treat 2 to 4 minutes with the following mordant: tannic acid 10 or 20%, 50 cc.; ferric chloride 5%, 10 to 15 cc.; carbol fuchsin (Ziehl-Nielson), 5 cc.; hydrogen peroxide 3%, 6 to 8 cc. Wash and stain 2 to 3 minutes with a mixture of basic fuchsin, saturated alcoholic, 10 cc.; anilin oil (1 part) and 95% alcohol (3 parts) mixed, 5 cc.; distilled water, 30 cc.; acetic acid, 4%, 1 cc. Wash thoroly with water.     

An improved staining technique for precipitin bands in agar or agarose gels

A method for the staining of proteins in agar and agarose gels using three stains simultaneously and a mordant is described. When compared with conventional Coomassie brilliant blue R-250 staining procedures, it requires a comparable time expenditure but has the following advantages: 1) it is threefold to fourfold more sensitive; 2) there is increased photographic resolution on conventional photographic material; and 3) the stain has a long shelf-life and does not fade under normal lighting conditions. Conditions for the washing and drying of gels are discussed.     

Nuclear staining with alum hematoxylin

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.     

An Evaluation of Cresyl Echt Violet Acetate as a Nissl Stain

The new cresyl echt violet acetate, of which three different batches have been tested, proves to be a very useful Nissl stain. It is especially valuable for formalin-fixed, frozen-sectioned material. By using a buffered staining bath and controlled timing in dehydration it is possible, on paraffin embedded material, to use these dyes as progressive stains apparently specific for nucleic acids. With a saturated aqueous solution of the dye, especially when a mordant of lithium carbonate is used, it is possible to stain material that has been preserved in formalin for several years and also material from which nucleic acids have been removed. The dye is useful also for staining celloidin embedded material. With the buffered stain proposed, differentiation is much easier than with older methods which included a gross overstaining and a long destaining procedure.     

Acceleration of the Movat Pentachrome One Stain by Heat

The conventional staining time for Movat's pentachrome I stain (Arch. Path., 60: 289-295, 1955) was shortened from about 18-19 hr to about 2.5 hr. The ammoniated alcohol and the resorcin-fuchsin staining baths were heated to 56 C. All other steps in the technic were performed at 25-27 C. Staining properties of paraffin sections of many types of tissue fixed in formalin, formol-sublimate-acetic, or in Bouin's fluid, showed that staining with resorcin-fuchsin at the elevated temperature gave the same results as staining at room temperature.     

The Cationic Chelate of Chromium and Aluminon as a Selective Nuclear Stain

The chelate k prepared by adding 4.5 gm of aluminon and 100 gm of chrome alum to 200 ml of distilled water, boiling gently for 20 min., filtering, and allowing the filtrate to drop into 3.5 liters of absolute alcohol. The alcoholic suspension is filtered and its precipitate is dried at room temperature. To prepare the staining solution 3 gm of chelate are dissolved in 100 ml of 3% HCI. Hydrated sections—paraffin, frozen, or celloidin—are stained for 30 min to 18 hr at room temperature. The stain is self-limiting and requires no differentiation. Since the stain is not removed by alcohol or weak acids, a large variety of counterstains my be used.     

Electron microscopy and X-ray microanalysis of a halophilic leptospire

The morphology of cells of strain Muggia, a slightly halophilic leptospire, was examined by the negative staining technique.The ultrastructure of the cells was rather similar to that of cells of Leptonema illini, i. e. the cells possessed cytoplasmic tubules. The basal complex of their flagella, however, was similar to the corresponding part of flagella on Gramnegative bacteria. The interior of the cells was densely packed with inclusions, except for the two outermost wavelengths at each end where these inclusions were absent.X-ray microanalysis showed that the inclusions contained sodium and chlorine as their main constituents. The inclusions disappeared upon storage of the cultures at room temperature.     

A simple, rapid method for demonstrating bacterial flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

Rapid Staining of Nuclei and Chromosomes in Root Tips

Feulgen reagent quickly heated to and maintained at 60 C just before immersion of plant material, basic fuchsin in acid alcohol at room temperature, and pinacyanol at room temperature will stain hydrolyzed root tip nuclei and chromosomes in one minute or less. This technic, coupled with fast fixation, can be utilized when uncertainties exist as to when to begin sampling plant meristem cells for mitoses or when time does not allow for standard fixation and Schiff staining.     

Control of the Ferric Ion Concentration in Iron-Aceto-Carmine Staining

The Fe⁺⁺⁺ concentration is controlled by adjusting the FeCl₃ normality of the iron-aceto-carmine staining solutions. Two stock mordant solutions are prepared by dissolving ferric chloride (FeCl₃·6H₂O; m. w. =270.31) in 45% glacial acetic acid, the normality of one being N/1, and of the other N/10. By combining aceto-carmine (preferably prepared from Merck's carmine No. 40 N. F., or from the Coleman and Bell product) and one or the other of the stock mordant solutions, a series of iron-aceto-carmine solutions is made up, each solution being of different normality for FeCl₃, depending on the proportions combined. Trial series: N/50, N/100, N/500, N/1000 and 0 N.

Tissue (spermatogenic) from one specimen is fixed in Carnoy-2, divided equally among the five iron-aceto-carmine solutions for staining, then squashed, dehydrated and mounted as usual. Subsequently the trial series may be retained or adjusted. Advantages of the method: 1) discloses quickly the optimum stain for a particular tissue type; 2) automatically gives an optimum stain to cells in different maturational stages; 3) results are reproducible in subsequent operations.

Tables and equations are provided for a number of other normalities and quantities of stain.     

A Simple, Rapid Method for Demonstrating Bacterial Flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

Nuclear stains with soluble metachrome metal mordant dye lakes

Summary Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe²⁺ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO₄ sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control.Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe²⁺ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results.Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes.The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes¹. Benzil at pH 13, which prevents the -naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.Assisted by Contract Nol-CB-43912 National Cancer Institute     

Application of stains-all staining to the analysis of axonemal tubulins: identification of beta-tubulin and beta-isotubulins.

The cationic dye, Stains-all, is known to stain brain beta-tubulin blue and alpha-tubulin red (Serrano, L. et al. (1986) J. Biochem. Biophys. Methods 12, 281-287; Serrano, L. et al. (1989) Biochem. Int., 19, 235-246). The present experiments show that this stain can also be applied to detect beta-tubulin in axonemal tubulins from various sources such as cilia of protozoa, sperm flagella of echinoderm, and sperm flagella of mollusc. Furthermore, these experiments showed that it selectively stains isoforms of axonemal beta-tubulin blue following isoelectric focusing, whereas those of alpha-tubulin are stained red. These results indicate that Stains-all staining is a useful tool for electrophoretic analysis of axonemal tubulins.