Neutral Red Dye Research Articles

Effects of concanavalin a and pokeweed mitogen in vivo on mouse peritoneal macrophages

Concanavalin A (ConA) and pokeweed mitogen (PWM) were injected intraperitoneally into mice and the effects on peritoneal macrophages were evaluated. When compared with saline injected controls, both mitogens caused at 4 days following injection a two-fold increase in the yield of macrophages, and these macrophages spread more rapidly on glass. Also, a higher percentage of heat-killed Candida albicans ingested by these macrophages stained red with neutral red dye. ConA, in contrast to PWM, produced a rapid, transient fall in macrophage yield within the first 3 h after injection. This effect could not be attributed to a direct agglutinating activity of the mitogen. Furthermore, only ConA resulted in the appearance of macrophages with an increased ability to phagocytize rabbit red blood cells in a serum-free medium.

Vital Staining to Sort Dead and Live Copepods

Intra vitam staining with neutral red dye vividly stains live copepods, providing a rapid method of sorting dead copepods from live. Organisms stained and preserved in acidic media retain coloration for several days, allowing large numbers of organisms to be leisurely sorted following bioassays. Cooling the preserved samples increases color retention. Copepods and cladocerans filter and ingest carmine particles providing an indication of feeding activity. Staining with neutral red at concentrations of 3:200,000 to 1:200,000 is relatively independent of temperature over a range of 5 to 30 C.

Supravital study of guinea pig cochlea referring especially to kanamycin labyrinthotoxy.

The organ of Corti has been studied supra-vitally with neutral red vital dye under normal conditions and during kanamycin intoxication. The absorption of the vital dye is described for both circumstances. Normally the living cell absorbs the substance in question as intraprotoplasmic granules, in large numbers. The inner hair cells take up the dye with greater intensity. The devitalized cells do not absorb it, or else do so in a diffused manner. In animals intoxicated with kanamycin, lack of absorption is observed in the outer hair cells, rarely in the inner ones; furthermore, such phenomena are more pronounced in the lower turns of the cochlea. The reason for this phenomenon is discussed, pointing out the possibility that it might be due to possible anoxia caused by the drug.

Observations on the plaque assay of visna virus.

Visna virus is the cause of a slowly evolving neurological illness of sheep (Sigurdsson, Pálsson & Grimsson, 1957; Sigurdsson & Pálsson, 1958). The virus has been propagated in cell cultures derived from sheep choroid plexus and quantified by 50% endpoint determinations of cytopathic effects in sheep choroid plexus cells 14 to 21 days after inoculation (Sigurdsson, Thormar & Pálsson, 1960; Harter & Choppin, 1967b). Attempts to obtain a plaque assay for visna virus have been hampered by the poor affinity which sheep choroid plexus cells have for neutral red and other vital dyes. To avoid this difficulty, an assay involving the development of plaques in a secondary cellular overlay, which could be stained with neutral red, was developed (Harter & Choppin, 1967a). An infected sheep choroid plexus monolayer of cells was maintained for 12 to 14 days under a semisolid overlay containing carboxymethylcellulose; the overlay was then removed and the sheep choroid plexus cells were covered with a sufficient number of baby hamster kidney cells (BHK 21-F) to establish a second confluent monolayer.

Method for Recovery of Viruses from Milk and Milk Products

A modification of the plaque-forming unit assay was developed for recovery of viruses from milk and milk products. Viruses were assayed on primary cell cultures of Cercopithecus aethiops (African green) monkey kidneys under an overlay medium. The medium contained 0.95% clarified Ionagar no. 2, Eagle's minimum essential medium with nonessential amino acids in Hanks’ balanced salt solution without phenol red, 2% fetal bovine serum, 0.19% NaHCO3, 0.0015% neutral red dye, 0.51% MgCl2 · 6H2O, and 1% sterile homogenized bovine milk. Geometric means and 95% confidence limits expressed as plaque-forming units/0.5ml are presented for recoveries of Adenovirus 12, Herpes simplex virus, Influenza A virus, and New castle disease virus from Eagle's medium and sterile milk. Recoveries of Adenovirus 12, Herpes simplex virus, Simian virus 40, and Reovirus 1 from milk, ice eream mix, chocolate milk, chocolate drink, or heavy cream were within±threefold of viral plaque-forming units input, estimated by dilution. Elimination or reduction of bacterial and fungal contamination in the nonsterile milk or milk products was achieved by adding 1,000 units of Penicillin G per milliliter, 1,000μg of streptomycin sulfate per milliliter, 50μg of tetracycline hydrochloride per milliliter, and 0.5μg of Amphotericin B per milliliter at least 48 hours before assay.

The disruption of lysosome-like particles of Solanum tuberosum cells during infection by Phytophthora erythroseptica Pethybr.

SUMMARY: Histochemical methods showed that tuber tissue and tissue culture cells of Solanum tuberosum contained phase-dense particles which absorbed neutral red and fluorescent dye, and particles which contained acid phosphatase and a non-specific esterase. It is suggested that these structures may be comparable to the lysosomes of animal tissues and the lysosome-like structures of plant cells which possess similar and additional properties. During infection of Solanum tissues by Phytophthora erythroseptica there was swelling and disruption of these host cell particles, accompanied by the release of acid phosphatase and esterase. Biochemical assay for acid phosphatase confirmed that infection of host cells resulted in the liberation of acid phosphatase from a particulate to the supernatant fluid fraction of cell homogenates.

The fine structure of pancreatic acinar and hepatic parenchymal cells after neutral red dye injection

The ultrastructure of mouse pancreatic acinar and hepatic parenchymal cells was studied sequentially after subcutaneous injections of neutral red dye. In both cell types, the develop- and control animals were noted. The pancreatic acinar and hepatic parenchymal ultrastructure was normal.

Studies on Echinostomatidae (Trematoda) in Malaya. XIV. Body Gland Cells in Cercariae of Echinostoma audyi Lie and Umathevy, and E. lindoense Sandground and Bonne

Cercariae of Echinostoma audyi and E. lindoense have, in addition to cystogenous and penetration gland cells, other gland cells that open on the body surfaces. These gland cells may be demonstrated by their intense staining with Nile blue sulfate, neutral red, and Bismarck brown. They also stain with brilliant cresyl blue, crystal violet, thionin, and toluidine blue. These para-esophageal gland cells, whose function is unknown, are absent in cercariae of Echinostoma malayanum, E. hystricosum, Echiroparyphium dunni, and Hypoderaeum dingeri, but present in cercariae of an Echinostoma with 37 collar spines, obtained from a Brazilian Biomphalaria glabrata. Cercariae of E. audyi and E. lindoense, though closely related, are easily differentiated by the distribution of these para-esophageal gland cells and their outlets. Lie and Umathevy (1965) indicated that mature, free-living cercariae of Echinostoma audyi Lie and Umathevy, 1965 and E. lindoense Sandground and Bonne, 1940, have, in addition to cystogenous and penetration gland cells other unknown gland cells that open on the body surface and stain intensely in dilute Nile blue sulfate or neutral red. This paper presents further details about these unknown gland cells. Because of their location near the esophagus I propose to name them para-esophageal gland cells. MATERIALS AND METHODS Cercariae of E. lindoense were obtained from experimentally infected Gyraulus convexiusculus (Hutton) and those of E. audyi from Lymnaea rubiginosa (Michelin). The supply of these cercariae was unlimited as the parasites were maintained in the laboratory. Living cercariae were stained with Nile blue sulfate, neutral red, and other intravital dyes diluted in half-strength physiologic saline and examined under oil immersion. Lillie's (1954) periodic acid-Schiff sulfite leucofuchsin (PAS) stain was employed for cercariae fixed in 5% formalin, 70% and absolute alcohol. Received for publication 25 May 1966. * This work was supported by the University of California International Center for Medical Research and Training (Hooper Foundation, San Francisco School of Medicine) with Research Grant GM 11329 from the Office of International Research, NIH, U. S. Public Health Service and by grant No. MSC-Hooper Foundation-02-Trevey Fund from the University of California School of Medicine, Committee on Research, San Francisco Medical Center. Cercariae of Schistosoma mansoni Sambon, 1907 were used as controls.

Further observations on the secretory cells in the optic tentacles of Helix, with special reference to the results of vital staining

ABSTRACT When the collar cells or the lateral cells in the tentacles of Helix aspersa are stained with neutral red and other vital dyes, uptake of the dyes occurs chiefly in the lipoidal β-bodies. Janus green B and dahlia are taken up by the mitochondria. In vitally-coloured preparations, no ‘peri-nuclear bodies’ are present in the collar and lateral oval cells, such as were previously found in these cells after post-osmication for 14 days. Fixed preparations of similar cells, post-osmicated for different periods, indicate that the peri-nuclear dictyosomes are artifacts due to over-impregnation with osmium. In addition to the lateral oval and lateral processed cells, which lie at intervals along the inner surface of the dermo-muscular sheath in Helix, unicellular calcium glands are found, lying immediately beneath the epithelial surface; these are far more numerous in H. pomatia than in H. aspersa. They contain granules of a calcium salt suspended in a matrix of mucopolysaccharide, and apparently produce the whitish mucoid secretion that is extruded on to the surface of the tentacles when the animal is disturbed. There is no evidence to suggest that the lateral cells are concerned in the production of this mucoid secretion.

Observations On Gut ph aNd Absorption of Methyl Red and Neutral Red in the Intestinal Walls of Pelodera and Mesodiplogaster

The literature on gut pH in nematodes is reviewed; early work was on free-living marine nematodes and later, on animal parasites. Except for Ascaris lumbricoide.r and Strongylus edentatus the nematodes tested showed acid gut reactions. The free-living soil nematodes, Pelodera lambdiensis and Mesodiplogaster sp. had oesophageal and intestinal contents that were just acid and the granular contents of cells of the intestinal wall were acid to different degrees. In Pelodera, cells just behind the anterior tip of the intestine were usually most acid. Methyl red and neutral red pH indicator dyes were absorbed selectively by different regions of the intestinal wall and by the two species.

Drinking by Adult Cephenemyia (Diptera: Oestridae)1

Frequent citations in the literature indicate that all adult bot flies are aphagous and without functional mouthparts. This paper describes imbibing of free water by two species of adult Cephenemyia . While in the process of drinking, the functioning of the external mouthparts was viewed through a stereoscopic microscope. Intake of fluids was confirmed by vivisection of flies offered a 0.01% solution of aqueous neutral red dye. Amount of fluid imbibed was measured by weighing flies immediately before and after drinking. The longevity of flies drinking water was significantly longer than that of the controls. Possible intake of free fluids by these flies under natural conditions is discussed, and a description of the anatomy of the mouthparts is presented.

Activation of corneal stromal cells to take up the vital dye neutral red

1. 1. A procedure is described for producing in vitro the activation of corneal stromal cells to transport the vital dye neutral red. The amount of dye taken up by the corneal stromal cells was quantitated by extraction of the dye from the cornea and measurement of the amount of dye extracted. 2. 2. Normal corneal stromal cells do not take up the dye, but cells activated by injury concentrate the dye approximately 150–250-fold. 3. 3. The transport of neutral red by corneal stromal cells may be broken down into 2 steps: ( a) activation of the corneal stromal cells to transport the dye, and ( b) the actual transport of the dye by the activated cells. 4. 4. Some factor (or factors) given off by the epithelium appears to be necessary for the activation of the cells. If the epithelium is removed atraumatically activation of the cells does not occur and dye is not taken up. In the presence of mildly injured (by cutting) epithelium, activation occurs during the 4th post-injury hour. Severe injury, produced by removing the epithelial cells by crushing them, leads to activation within 1 hour. Direct trauma to the corneal stromal cells in the absence of the epithelium does not activate them to transport neutral red. 5. 5. Proteolytic activity in the injured epithelium apparently leads to the liberation from the epithelium of the factor (or factors) which activates the corneal stromal cells. Soybean trypsin inhibitor prevents activation of the corneal stromal cells in the presence of epithelium but not in the absence of epithelium (removed traumatically). Trypsin is an effective activator of the neutral red transport by corneal stromal cells. Trypsin, however, causes the reaction only in the presence, but not in the absence, of epithelium.

On the excretory function of the stomach

The effect of food and histamine on the dynamics of gastric excretion of urea, ammonia and neutral red dye was studied in ten dogs with permanent Basov flstulse, and two dogs with Pavlov pouches. No correlation between the excretion of urea, ammonia, neutral red dye and the intensification of gastric secretion was noted. The amount of substances excreted by the stomach depended mainly on their concentration in the blood.

The Cellular Reaction in Experimental Syphilis. Supravital and Fixed Material.

The blood and tissues of rabbits inoculated with Treponema pallidum have been studied from the standpoint of the cellular reaction at different periods of the disease. The evidence from successive weekly blood counts on 5 groups of rabbits, 40 animals in all, indicated that the various classes of cells were numerically affected. A striking finding was the increase in the numbers of circulating monocytes associated with actively developing lesions. The increase was very much more pronounced than the spontaneous rises of monocytes noted in groups of normal rabbits, the blood of which was examined at similar intervals over prolonged periods of time. Similar experiments have been carried out in which various tissues involved by the disease process were examined, both in supravital and in fixed preparations, in order to study the relationship between the blood picture and the types of cells concerned in the tissue reaction. Supplementary observations were also made on a large number of syphilitic rabbits in which the blood was not examined systematically; every instance of tissue examination, however, was immediately preceded by a blood count. Standardized pipettes were used and the differential counts were made with the supravital technique. Scrapings of fresh tissue were examined by the supravital method, using neutral red and Janus green dyes, and fixed preparations of the tissues were made with various fixatives and stains. In all experiments, special attention was paid to the clinical character of the disease. The particular lesions studied and the time selected for their examination was largely determined by the general course of the infection, and it should be emphasized that an essential feature of the study was the examination of lesions at different stages of development.

The Hydrogen Ion Concentration of the Nucleus and Cytoplasm of the Egg Cell.

By means of the microinjection apparatus the series of acid dye indicators of Clark and Lub and the basic dye Neutral Red were injected both into the cytoplasm and into the nucleus of the immature starfish egg. The color changes upon injection indicate the pH of the nucleus to be in the neighborhood of 7.6 to 7.8, whereas that of the living cytoplasm is 6.6 to 6.8, and cytoplasm cytolyzed by mechanical injury is 5.4 to 5.6.

GASTRIC EXCRETION OF NEUTRAL RED

The application of neutral red as a vital stain has been a wide one since its introduction by Ehrlich into medicine. Its use in the study of secretory functions dates from Fuld's 1 work on dogs with Pawlow pouches in 1908, in which he demonstrated its excretion into the pouch, when the dye was instilled into the main stomach. Finkelstein, 2 in 1922, injecting neutral red and other dyes subcutaneously, studied the excretion into Pawlow pouches and substantiated Fuld's earlier observations. Glaessner and Wittgenstein, 3 in 1923, applied this knowledge to the formulation of a gastric function test, using as their criterion the appearance time of the dye in the stomach after intra-muscular injection. Subsequently, publications by Simici and Dumitriu, 4 Koopman, 5 and Carnot and Gaehlinger 6 have emphasized the usefulness of this procedure. The studies reported here aim particularly to consider the clinical usefulness of determining the