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When staining the internal phloem region of a potato tuber with the vital stain neutral red, it was observed that files of elongated cells of narrow diameter were heavily stained and were easily distinguishable from the more isodiametric parenchyma cells, many of which did not stain with neutral red. The elongated cells were identified as companion cells by locating the adjacent sieve-tube members through counterstaining with aniline blue and reviewing under violet light. Of a number of other plants surveyed, only parsnip roots possessed companion cells exhibiting a similar slective staining. In other plants both the companion cells and the surrounding parenchyma cells usually stained. Sieve-tube members never accumulated neutral red. It was concluded that the vacuoles of the companion cells of the potato tuber were stained by the ion trap mechanism because of the color of the accumulated stain, the lack of staining when neutral red was applied in an acidic solution, and the complete destaining after soaking in dilute ammonium hydroxide.

Mouse cells cultivated in vitro were continuously labeled with tritiated thymidine for 48 hr. In representative groups of labeled cells, the percentages of dead cells were obtained by vital staining with either neutral red or trypan blue. After fixation and auto-radiography, the fraction of nonlabeled cells was determined in the same group of cells which had been used for interpretation of viability. Neither neutral red nor trypan blue used prior to autoradiography caused spurious grain formation in the emulsion.

Neutral red iodide suitable for vital staining was prepared by condensing nitrosodimethylanilin hydrochloride with m-toluylenediamine and the indamine, toluylene blue, was obtained. This was subjected to air oxidation and converted to the eurhodine, neutral red. The purification of this dye was brought about by converting it into its comparatively insoluble stannous chloride double salt, filtering, dissolving in water, and precipitating the neutral red iodide with potassium iodide solution. This was re-dissolved in water, reprecipitated with potassium iodide solution and crystallized from 95% ethanol. The uncrystallized dye was also found satisfactory for vital staining. Several other preparations of neutral red iodide were made, using a somewhat different procedure than that given above, and it was generally found that satisfactory stains were obtained only when the preparation was free from toluylene blue.The chloride of the color base was prepared by continuing the air oxidation of the toluylene blue u...

Trypan blue granules within the histiocytes and neutral red stained granules within the mast cells of the same areolar tissue spread can be preserved in their original colored state by the following method: fix in 10% formalin for 12–24 hours; rinse in distilled water; place in two changes of dioxan for 5–10 minutes each (overnight in second change will not hurt); mount from the second dioxan using diaphane dissolved in dioxan as a mounting medium, or clear in xylene and mount in balsam. No dye is lost, and no cellular distortion occurs. Fast green is used as a counterstain (1% aqueous plus 0.2% acetic acid). Stain ½ to 1 minute and rinse.

Aqueous 0.1% neutral red adjusted to pH 6.5, or mixed 1:1 with seawater, produced conspicuous fluorescence in plant and animal fats while acting as a conventional stain for acidic groups including volutin. The water-soluble, nonfluorescent dye predominating in acid solutions became deprotonized, lipophilic and fluorescent under neutral and alkaline conditions. Neutral dye crystals fluoresced dull red, whereas the fluorescence was either bright lemon yellow or blue-green in lipids, apparently dependent upon their composition.

Modifications of the neutral red vital staining technique of Drgsel and co-workers to sort live and dead marine copepods have made possible live-dead determinations of most components of estuarine ...

(1927). The Preparation of Neutral Red Iodide Suitable for Vital Staining. Stain Technology: Vol. 2, No. 1, pp. 17-18.

Exposure of three typical organisms—Serratia marcescens Bizio (syn. Bacillus prodigiosus), Bacillus anthracis Koch, and Staphylococcus aureus Rosenbach—to saturated aqueous solution of neutral red (5.6% National Aniline certified) for 48 hours failed to kill them; and Serratia marcescens survived 9 days exposure. All three organisms survived 21 days exposure to 2% solution of neutral red and Bacillus anthracis survived thirty days exposure to this dilution of dye. The three organisms survived 24 hours exposure to saturated aqueous Bismarck brown (Grubler) and Bacillus anthracis survived 40 days exposure. Serratia marcescens (known to be one of the Gram-negative group resistant to triphenylmethane dyes) was tested for degree of resistance to gentian violet. The bacteriostatic titre of this dye in agar for this organism is about 1: 900. It was found to survive 4 hours exposure to 1:2000 solution and is probably unaffected by even much stronger dilutions. To aqueous suspensions of this organism enough gentia...

Several samples of neutral red have been studied; first, for the determination of their relative color intensities, and second, to determine their toxic effects on Paramoecium caudatum.It was found that each of these dyes varied in physical characters both in dry form and in solution. The duration of life of groups of Paramoecia varied in each dye solution. These differences are probably due, at least in part, to differences in the chemical composition of each dye. This suggests that the varying results obtained by experimenters, in staining living material when using the same kind of dye under uniform conditions, may probably be attributed to these differences of chemical constitution of the dyes.In the comparison of the toxic effects of the several samples most uniform results were obtained at concentrations of 1:5,000 and 1:10-000. At a concentration of 1:100,000 neutral red was found toxic but the results obtained were not in agreement with those obtained at lower concentrations.

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 x magnification). The fluorochrome is seen to be accumulated in the liposomal membranes. Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time. Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.

There are few histochemical techniques for staining rubber in the cells of guayule. With regard to specificity, clarity and retention of the stain we found staining with oil red O or iodine-potassium iodide for bright field, and a combination of oil red O and dansyl chloride for epifluorescence microscopy to be the best methods. The plausible mechanisms of staining are discussed.

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 microns. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.

Aqueous solutions of the arylmethane dyes Chromoxane pure blue BLD (C.I. No. 43825) and Chromoxane pure blue B (C.I. No. 43830) will stain beryllium oxide. In the presence of EDTA the staining of other metals is masked. As a specific stain for BeO, formol saline fixed paraffin sections are hydrated and stained for 1 hr with either 0.1 gm of pure blue BLD in 100 ml of pH 4.0 Na-acetate buffer or with 0.1 gm of pure blue B in 1 N NaOH adjusted to pH 9.0 with HCl. To mask interference from other metal ions, 9 gm of Na2-EDTA is added to 100 ml of the stain solution. BeO is stained blue, organic tissue components are either unstained or pink. Results of tests against other materials show that a high degree of specificity may be expected from these dyes. A 1% aqueous solution of neutral red may be used as a counterstain.

The cells were smeared in water or water which had stood over about 10 mg. of magnesium powder per ml. for 30 minutes or longer. After the smear was dry and whitish in appearance it was held over a beaker of hot water (60-65° C.) until it was translucent or becoming translucent and exposed immediately to hydrogen chloride (gas) for a few seconds. After drying, it was covered with a 0.1% aqueous solution of neutral red for 5-8 minutes. The excess stain was washed from the slide with water and, while wet, placed in a saturated aqueous solution of mercuric nitrate for 5-15 seconds. The smear was rinsed in water and allowed to dry. When dry the slide was placed on a 50° C. warm plate and covered with a thin film of a 5% aqueous solution of nigrosin adjusted to a pH of about 3. The film dried quickly and upon cooling was ready for study. The stained material in the cells varied in shape and location with the moisture content of the smear and the time of exposure to hydrogen chloride. In the area of the smear d...

Pinacyanol1 is an excellent, almost permanent and very selective stain for mitochondria in supra-vital blood preparations. It may be used alone or in combination with neutral red for the study of fresh blood, bone marrow, spleen or lymph node smears. One drop of a 0.1% solution of pinacyanol in absolute alcohol, per cubic centimeter of the same solvent, is a satisfactory dilution for making stain films for blood with an average concentration of cells.