Neutral Red Vital Staining Research Articles

Lysosomes, caspase-mediated apoptosis, and cytoplasmic activation of P21, but not cell senescence, participate in a redundant fashion in embryonic morphogenetic cell death

Micromass cultures of embryonic limb skeletal progenitors replicate the tissue remodelling processes observed during digit morphogenesis. Here, we have employed micromass cultures in an in vitro assay to study the nature of cell degeneration events associated with skeletogenesis. In the assay, “naive” progenitors obtained from the autopod aggregate to form chondrogenic nodules and those occupying the internodular spaces exhibit intense apoptosis and progressive accumulation of larger cells, showing intense SA-β-Gal histochemical labelling that strictly overlaps with the distribution of neutral red vital staining. qPCR analysis detected intense upregulation of the p21 gene, but P21 immunolabelling showed cytoplasmic rather than the nuclear distribution expected in senescent cells. Semithin sections and transmission electron microscopy confirmed the presence of canonical apoptotic cells, degenerated cell fragments in the process of phagocytic internalization by the neighbouring cells, and large vacuolated cells containing phagosomes. The immunohistochemical distribution of active caspase 3, cathepsin D, and β-galactosidase together with the reduction in cell death by chemical inhibition of caspases (Q-VAD) and lysosomal cathepsin D (Pepstatin A) supported a redundant implication of both pathways in the dying process. Chemical inhibition of P21 (UC2288) revealed a complementary role of this factor in the dying process. In contrast, treatment with the senolytic drug Navitoclax increased cell death without changing the number of cells positive for SA-β-Gal. We propose that this model of tissue remodelling involves the cooperative activation of multiple degradation routes and, most importantly, that positivity for SA-β-Gal reflects the occurrence of phagocytosis, supporting the rejection of cell senescence as a defining component of developmental tissue remodelling.

Bivalve Oocyte Inviability Revealed Using a Simple Staining Technique

Photo 1. Cerastoderma edule (Linnaeus, 1758). (A) Emerged animals at low tide, (B) submerged animals with foot (F) and inhalant (IS) and exhalent (ES) siphons deployed. Animals in (A) and (B) are in their 3rd year (two annual growth rings are visible, corresponding to periostracal thickening) and thus both sexually mature. (C) Oocytes (OC) double-stained with Neutral Red vital stain and Trypan Blue mortal stain. Left, normal oocyte, right, dead oocyte. TB, undissolved Trypan Blue stain. (D) Oocytes stained with Neutral Red only; normal oocytes (NO) stain red, while dead oocytes do not stain. The TB stain proved effective, but cumbersome and costly to use in routine studies, while the more convenient and cost-efficient Neutral Red stain alone provided the same information. Photo credit: D. Chérel. These photographs illustrate the article “Evolutionary and ecological insights from vital staining of bivalve oocytes: A Red Queen at the sweepstakes?” by Peter G. Beninger, Daphné Chérel, Gaël Le Pennec, and Sandra E. Shumway published in Ecosphere. https://doi.org/10.1002/ecs2.4047

Evolutionary and ecological insights from vital staining of bivalve oocytes: A Red Queen at the sweepstakes?

AbstractTo determine the proportion of inviable oocytes that are spawned along with normal oocytes in marine bivalves, we turned to a time‐honored technique not previously used on invertebrate oocytes: the neutral red vital stain. Despite the difficulty in obtaining induced spawns from field‐collected Cerastoderma edule (L), the results and insights from this simple procedure to answer a simple question were unexpected and far‐reaching. Mean proportions of inviable oocytes in the spawns of cockle C. edule ranged from 35% to 85%, with the vast majority represented by atresic (autolyzing) oocytes. These levels are in agreement with previous quantitative histological observations of prespawning atresia in both C. edule and the Manila clam Tapes philippinarum. Such elevated prefertilization inviability mirrors the known high level of postfertilization inviability in bivalves. Together, these high levels of inviability may account for much of the early mortality in the type III survivorship curves typical of marine bivalves. The combination of high fecundity and high oocyte inviability suggests a Red Queen‐type reproductive dilemma in these species. In addition, the great degree of interindividual variation observed in oocyte inviability suggests that cockles, and perhaps other intertidal bivalves, present a Sweepstakes Reproductive Strategy, in which highly variable individual fecundity confronts a highly variable environment. The neutral red technique promises to be a useful tool for investigating prespawning inviability in other marine taxa, as well as for aquaculture, conservation, restocking, ecophysiology, and environmental monitoring.

Examining bivalve fecundity: oocyte viability revealed by Neutral Red vital staining

Estimation of realized fecundity (Freal, number of viable oocytes produced) is an essential, yet seldom-achieved element in the understanding of marine animal production and population dynamics. We used the Neutral Red (NR) vital stain to determine oocyte viability in spawns and gonad strippings of four species of commercially-important bivalves: Cerastoderma edule (L), Crassostrea gigas Thunberg, Mytilus edulis L, and Tapes philippinarum (Adams and Reeve). The utility of Trypan Blue as a complementary mortal stain was also assessed, and found to be unnecessary. Normal, live oocytes stained with NR, and were either spherical (mature oocytes) or pedunculated (immature oocytes); atresic/abnormal oocytes also stained with NR, allowing their ready recognition due to their abnormal shapes and cytoplasmic retraction. Dead oocytes did not stain with NR. Across the species studied, a considerable and highly variable proportion of spawned or stripped oocytes was either dead or non-viable; quantitative counts were performed for Cerastoderma edule, the only species for which > 5 spawns occurred. The high level and variability (34–85%) of dead or non-viable oocytes is consistent with a reproductive Red Queen dilemma, in which greater oocyte numbers do not translate to commensurately greater real fecundities, and also with a Sweepstakes Reproductive Success strategy, in which a large range of Freal confronts the considerable variability of intertidal environmental conditions. Neutral Red vital staining is a promising tool for the elucidation and optimization of crucial yet previously intractable aspects of bivalve hatchery production, genetic improvement, restocking, stock management, and conservation.

Inhibition of cytotoxicity of Shiga toxin of Escherichia coli O157:H7 on vero cells by Prosopis alba Griseb (Fabaceae) and Ziziphus mistol Griseb (Rhamnaceae) extracts.

The capacity of Prosopis alba Griseb. and Ziziphus mistol Griseb. fruit extracts to inhibit the toxic action of Shiga toxin (Stx) was investigated. Purification of Stx from Escherichia coli O157:H7 was performed by saline precipitation and affinity chromatography using a column with globotriaosylceramide, while the fruits were subjected to ethanolic or aqueous extractions. The protective action of both fruits was determined by pre-, co-, and postincubation of one 50% cytotoxic dose per ml of Stx with different concentrations of ethanolic and aqueous extracts in confluent monolayers of Vero cells for 72 h at 37°C (5% CO2). The inhibition of the cytotoxic effect of Stx by fruit extracts was determined by the neutral red vital staining technique. The extraction of the polyphenols and flavonoids was effective, and more polyphenols per milligram of dissolved solids were obtained from P. alba than from Z. mistol. However, there were more flavonoids in Z. mistol than in P. alba. Components of both fruits increased the viability of cells treated with Stx when the extracts were preincubated with Stx for 1 h before being applied to the cell cultures, with the ethanolic extract of P. alba showing 95% cell viability at a concentration of 2.45 mg/ml. The extracts were less effective in protecting cells when Stx, extracts, and cells were coincubated together without a previous incubation of Stx; only the concentrations of 19.46 mg/ml for the P. alba aqueous extract and 3.75 mg/ml for the Z. mistol ethanolic extract resulted in the inhibition of cytotoxicity, with 52 and 56% cell viability occurring, respectively. Investigation into this difference in the protection of cells indicated that the protein molecule of Stx suffered degradation to advanced oxidative protein products during preincubation with extracts, principally with P. alba, which exhibited a greater amount of nonflavonoid polyphenols than Z. mistol. The prooxidant action on Stx favored the cells and enhanced the protective action of both fruits.

Dead or alive? Viability assessment of micro- and mesoplankton

The rapid and efficient analysis of plankton samples (e.g. enumeration, identification, biomass determination) has been an important driver for recent technological developments in (semi-) automated analysis and imaging instruments. Most focus has been on identification and abundance estimates, while less attention has been given to viability, i.e. the assessment of whether the organisms are dead or alive. However, a wide spectrum of scientific applications requires accurate viability determinations, e.g. the monitoring of invasive species in ship ballast water. The transfer of species through ballast water forms a major threat to marine ecosystems, resulting in significant environmental and economic losses. A variety of viability stains and viability assessment methods are available, but there has been no systematic investigation how these methods perform for larger organisms (epsilon 50 m). We review the current procedures for viability determination for large plankton and present a cross-comparison of three methods: cell digestion assay (CDA), SYTOX Green nucleic acid staining and Neutral Red vital staining. The CDA and SYTOX Green methods did not perform well and gave various problems linked to the multicellular nature of zooplankton, autofluorescence and/or constraints set by the definition of cell death. Although some issues remain and there is no universal method, the Neutral Red vital stain proved the most robust viability method in this study and is broadly applicable to both phytoplankton and zooplankton larger than 50 m.

Intracellular alkalinisation in Vero cells parasitised by Trypanosoma cruzi

We studied the intracellular pH of Vero cells parasitised by Trypanosoma cruzi, using different methods: fluorimetric measurement after labelling the cells with the pH-sensitive intracellular fluorescent dye 2′,7′,-bis- (2-carboxyethyl)-5- (and-6)-carboxyfluorescein, acetoxymethyl ester; flow cytometry; and image analysis after staining the cells with neutral-red vital stain. The results show that the intracellular pH of the parasitised cells rose in comparison with that of the uninfected control cells. A study of the population of parasitised cells made by flow cytometry allowed us to subdivide the cells from the infected cultures into two populations according to their pH as obtained by fluorimetric measurements. Image analysis showed that the cell cytoplasm was more alkaline in the vicinity of the sites containing parasites. Treatment of the parasitised cells with amiloride, ouabain, or with 4,4′-diisothiocyano-2,2′-stilbene disulphate consistently lowered the pH values of the parasitised cells, but not sufficiently to return to the values of the non-parasitised control cells. When the control cells were subject to similar treatments with the inhibitors, only amiloride acidified the cytoplasm to any extent. The basification undergone by the parasitised cells was independent of the transport systems and may be a consequence of the release of NH + 4 by the intracellular amastigotes. © 1998 Australian Society for Parasitology.

The susceptibility of tissue cell walls toErwinia enzymes differs among the potato cultivars

In the cell wall matrix of higher plants, cellulose fibrils are embedded in a network of pectic substances, hemicelluloses and structural proteins. But plant pathogenicErwinia carotovora bacteria secrete a mixture of enzymes including pectinases, cellulases and proteases to degrade the cell walls of host plant tissue. Tuber tissue of 31 potato genotypes was exposed to a mixture ofErwinia enzymes before the degree of cell wall lysis was determined by a Neutral-red vital staining method. The data indicate that the resistance of cell walls to the action ofErwinia enzymes differed significantly among the potato cultivars and clones. The decrease in cell viability caused by the action of enzymes ranged from 7% for cv. Maxilla to >46% for cv. Arnika. An increased rate of N-fertilization coincided with enhanced susceptibility of cell walls to the action ofErwinia enzymes. There was a correlation between the amount of cell wall substances in tuber tissue and the resistance of cell walls to enzymic degradation.

Induction of renin release from isolated glomeruli by inorganic mercury(II)

Mercury(II) ions are known to accumulate in the kidney and their effect upon the renin-angiotensin system has also been described. The question, however, whether mercury(II) also exerts direct effect on the juxtaglomerular cells (JGC) to induce renin release remained to be answered. Suspension of isolated glomeruli was used to measure the mercury(II)-induced renin release in vitro. The glomeruli were isolated from female BALBc mice. HgCl 2 was found to be capable of inducing renin release directly from JGC. The effect is concentration-dependent ( P < 0.05, r = 0.914 and P < 0.01, r = 0.982, with and without Neutral Red vital staining) and becomes apparent already at a mercury(II) ion concentration as low as 1 μM. The renin-releasing effect of the mercury ion is to be inhibited by dithiothreitol (DTT) (renin activity 20.37 vs. 2.60 ng/ml.h in supernatant) as well as the elevated osmotic concentration of the incubating bath medium (20.37 vs. 6.84 ng/ml.h). This suggests that certain membrane sulfhydryl groups are implicated in the process on the one hand, and it is also in accordance with the known sensitivity of the renin granules to osmotic pressure on the other hand. Light and electron micrographs also demonstrate the direct, effective role of Hg(II) in the renin release process. Therefore, it is assumed that apart from its influence on tubulo-glomerular feedback a direct way of action of mercury(II) on renin release must also be taken into account.

Cytotoxicity of retrofill materials

Dentin bonding agents reduce microleakage and enhance marginal adaption of composite resin restorations. These characteristics are advantages for their use as an endodontic retrofilling material. Because these materials will be in direct contact with vital tissues, their cytotoxic potential must be evaluated before clinical use. It was the purpose of this study to evaluate the cell cytotoxicity of amalgam, Caulk Universal Bond, Gluma, 35% HEMA, Morita Clearfil, Scotchbond 2, Super EBA, Tenure, and Tenure 5-4. VERO cells were grown in RPMI-1640 medium and cell monolayers were prepared by incubating 15 ml of the cell suspension in 60-mm culture dishes at 37 degrees C in 5% CO2. Twelve milliliters of a medium-agarose mixture containing 1% neutral red vital stain were overlayed onto the cell layer and allowed to solidify. The materials were directly exposed to the agarose overlays by inverting 6.0-mm diameter polypropylene capsules containing the cured and liquid sample materials either immediately (0 time) or after placement in phosphate-buffered saline with 1% gentamicin for 7, 15, or 30 days. Cytotoxicity was determined by measuring the zone of killed cells around the sample 24 h after placement on the agarose. Cytotoxicity was determined by measuring the zones of cell inhibition at 24 h and at 7, 15, and 30 days. Initially, all of the materials were found to be cytotoxic, except amalgam and the Tenure components. The dentin bonding primers showed a mean zone of inhibition of 13.2 mm and the cleansers a 40.0-mm zone. Amalgam demonstrated increasing cytotoxicity: 0.0 mm at 24 h to 12.0 mm at 30 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Virulence and Enzyme Production of Erwinia carotovora ssp. atroseptica on Potato Tuber Tissue

AbstractThe elicitation of pathogenesis on potato tuber slices by 6 strains of Erwinia carotovora ssp. atroseptica was investigated by neutral red vital staining and has been compared with bacterial growth rate, penetration ability, enzyme production and enzyme spectrum. The induction of enzyme synthesis (particularly, of the extracellular polygalacturonase) elicites the rot attack on tuber tissue and this requires a sufficient bacterial density.Due to wound healing, the inductors of enzyme production are removed, and after 48 h enzymes do not attack tuber tissue any more. Therefore, growth rate and penetration ability (to get the necessary bacterial density and inductor substances) may limit virulence. A similar influence of enzyme production and enzyme spectrum of the strains on the virulence was not detected.

Interdigital tissue chondrogenesis induced by surgical removal of the ectoderm in the embryonic chick leg bud

In the present work, we have analysed the possible involvement of ectodermal tissue in the control of interdigital mesenchymal cell death. Two types of experiments were performed in the stages previous to the onset of interdigital cell death: removal of the AER of the interdigit; removal of the dorsal ectoderm of the interdigit. After the operation embryos were sacrificed at 10-12 h intervals and the leg buds were studied by whole-mount cartilage staining, vital staining with neutral red and scanning electron microscopy. Between stages 27 and 30, ridge removal caused a local inhibition of the growth of the interdigit. In a high percentage of the cases, ridge removal at these stages was followed 30-40 h later by the formation of ectopic nodules of cartilage in the interdigit. The incidence of ectopic cartilage formation was maximum at stage 29 (60%). In all cases, cell death took place on schedule although the intensity and extent of necrosis appeared diminished in relation to the intensity of inhibition of interdigital growth and to the presence of interdigital cartilages. Ridge removal at stage 31 did not cause inhibition of the growth of the interdigit and ectopic chondrogenesis was only detected in 3 out of 35 operated embryos. Dorsal ectoderm removal from the proximal zone of the interdigit at stage 29 caused the chondrogenesis of the proximal interdigital mesenchyme in 6 out of 18 operated embryos. The pattern of neutral red vital staining was consistent with these results revealing a partial inhibition of interdigital cell death in the proximal zone of the interdigit. It is proposed that under the present experimental conditions the mesenchymal cells are diverted from the death programme by a primary transformation into cartilage.

Syndactyly induced by Janus Green B in the embryonic chick leg bud: a reexamination

In an attempt to clarify the mechanism of production of the syndactyly induced by Janus Green B (JGB) we have studied the morphology and structural modifications of the chick embryo leg bud after JGB administration by means of neutral red vital staining, whole-mount cartilage staining and light microscopy and transmission and scanning electron microscopy. The results show that the well-known inhibition of interdigital cell death is accompanied by a precocious alteration of the epithelial tissue and especially of the epithelial-mesenchymal interface. 24 h after JGB administration the cells of the AER reduce the number of junctions and the basal ectodermal cells are detached into the mesenchymal tissue in zones in which the basal lamina undergoes disruption. In addition the interdigital mesenchymal cells diverted from the dying program are able to undergo a rapid differentiation into cartilage. It is proposed that the mechanism of production of JGB-induced syndactyly might be due to an alteration of the normal epithelial-mesenchymal interactions rather than to a direct inhibitory effect of the JGB on the dying program.

Cell death in the dorsal part of the chick optic cup. Evidence for a new necrotic area

The spatiotemporal pattern of morphogenetic cell death during the early development of the chick retina was studied by means of the neutral red vital staining and light microscopy. A modification of the conventional procedure of vital staining, which consisted of the injection of the dye into the neural tube lumen, was used for this purpose. In addition to the two areas of cell death known from previous literature, the first located in the ventral part of the optic cup and the second located in the insertion of the optic stalk with the diencephalon, a new area of cell death was described. This third necrotic area was located in the protruding dorsal part of the optic cup rim and was present throughout the stages 15 to 18. The area consisted of dying cells, fragments and phagocytosed cells. We suggest that this dorsal area of cell death could stop the intense dorsal growth of the optic cup and/or reshape the optic cup rim. Moreover, this area may influence the production of cell degeneration in the dorsal part of the invaginating lens placode.

Characterization of Orthophosphate Absorption by Pea Root Protoplasts

Root protoplasts were isolated from 4 d old seedlings of Pisum sativum. Viability was verified by fluorescin diacetate, direct blue and neutral red vital staining techniques. Phosphate influx of the protoplasts was similar to that of roots with respect to phase 1 affinity for orthophosphate (Km = 9-9 mmol m~3) and sensitivity to pH and metabolic inhibitors. Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone decreased uptake to 4% of the control and diethyl stilboestrol induced leakiness in the protoplasts. Influx was only slightly less than that in plasmolysed roots which in turn was 3-6 times lower than that of normal roots. Efflux from the protoplasts was high with a value of approximately 2% of the cellular phosphate content per hour. The elevated leakage indicated an essential difference between the effects of protoplast isolation on influx and efflux of phosphate.

Improved yields and assay of simian varicella virus, and a comparison of certain biological properties of simian and human varicella viruses

Studies were performed to define conditions under which propagation, assay and stabilization of the Delta herpesvirus (DHV) strain of simian varicella virus might be improved, and to compare biological properties of DHV with those of human varicella zoster virus (VZV). A mycoplasma contaminant was successfully eliminated from the DHV seed virus by treatment with a specific antiserum. DHV was found to replicate more efficiently in the BS-C-1 line of African green monkey kidney cells than in Vero cells, and seed virus preparations in the form of virus-infected cells were produced which had infectivity titers ⩾ 1 × 10 6 p.f.u./ml. Greater yields of virus were produced in cultures infected as dispersed cells than as preformed monolayers. Infectious DHV could be released from host cells by sonic treatment of heavily infected cultures at 48 h post infection. Certain agents reported to enhance replication of herpes viruses (caffeine, carbaryl, the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate, and DEAE-dextran) had no enhancing effect on replication of DHV. However, DEAE-dextran in the maintenance medium enhanced spontaneous release of DHV into culture fluids. Plaquing efficiency and plaque size of DHV were greater in BS-C-1 than in Vero cells, and plaque assays and plaque reduction neutralization tests were developed in this cell system using a solid overlay medium with neutral red vital stain. Neutralization of DHV was markedly enhanced by fresh guinea pig complement. The newly developed neutralization test demonstrated more vigorous antibody responses to DHV in active and latent VZV infections than were demonstrated with previous procedures. In addition to their preferential growth in monkey and human cells respectively, DHV and VZV were found to differ markedly in their rates of attachment to host cells, with DHV requiring over 6 h of adsorption, while VZV adsorption was essentially complete at 1 h. Also, cell-free DHV was much more resistant than cell-free VZV to repeated cycles of freezing and thawing.

Evaluation of biologic effects of dental materials using four different cell culture techniques

The cytotoxicity of fresh and 1-day-old silicate cement, composite restorative material and zinc oxide-eugenol cement (ZOE) was tested using human epithelial cells (NCTC 2544) in four different cell culture systems: (1) 51Cr-release from prelabeled cells after incubation for 4 and 24 h in the presence of the materials. (2) Implanting the materials on an agar everlay and visualizing any cytotoxic effects after 24 h by neutral red vital stain. (3) Cell growth during 5 d in the presence of the materials. (4) Colony-forming ability after exposure of the cells for 30 min to medium previously incubated with the materials for 24 h. Freshly prepared and 1-day-old ZOE exhibited a prominent cytotoxic effect in all four systems. A less marked effect was found for the composite material in systems 2, 3, and 4, while silicate cement appeared to be the least toxic material in these three systems. Neither silicate cement nor composite showed any cytotoxic effect in system 1 based on 51Cr-release. It is concluded that the effects obtained by the cell culture techniques did not mimic the reactions obtained when the materials are tested under conditions which reflect their clinical use.

The use of neutral red and Evans blue for live-dead determinations of marine plankton (with comments on the use of rotenone for inhibition of grazing).

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 zooplankton. A stock solution of neutral red is added to a zooplankton sample to give a final concentration of 1:100,000. The length of the staining period vanes from one to six hours, depending on which taxa live-dead determinations are to be made. Calanoid copepods, their nauplii and polychaete larvae require the shortest staining period; barnacle nauplii and cyprids, and decapod zoeas require the longest time. Evans blue, which stains dead diatoms blue but dm not stain living ones, gives satisfactory results for live-dead determinations of most estuarine diatoms in final concentrations of 1:2000 to 1:400. The diatoms Biddulphia and Melosira, and the dinoflagellate Peridinium stained inconsistently with Evans blue, but live-dead determinations were successful using neutral red in a final concentration of 1:50,000. Grazing is a problem in plankton samples held for live-dead determinations of diatoms. This problem was reduced by using rotenone in a concentration of 2.5 pm to kill the calanoid copepod component of these samples.

輸血用血液中の白血球系細胞の生存期間の検討

I studied on the survival time of leucocytes in whole blood preserved in acid citrate dextrose (ACD) solution at 4°C and found that mononuclear leucocytes were rapidly killed. These conclusions were based on the loss of mitosis in tissue cultures at 37°C during the 10 days of storage, and a decrease in survival rate in neutral red vital staining during the 14 days of storage.

簡易骨髄組織培養法に関する研究

With the use of the simple bone-marrow tissue culture, the method devised in our laboratory, in the medium composed of serum and vitamin B12, the author studied the neutral red vital staining of neutrophils in the bone marrow of both normal persons and patients with various blood diseases. These results were compared with those obtained by the conventional method using the medium composed of plasma mixed with heparin and chick embryo juice; and the author arrived at the following conclusions:1. The average stain of mature neutrophils in the case of normal persons reaches the maximum of 1.12 within 4 hours after the addition of neutral red solution, fading gradually thereafter.2. In the case of leukemia mature neutrophils in the bone marrow are stained more deeply at an early stage as compared with those of normal persons; and in the acute leukemia they fade at an early stage, and in the chronic leukemia they fade more slowly than those of acute leukemia.3. In the case of hypoplastic anemia mature neutrophils in the bone marrow are stained deeper and earlier than those is normal persons, and also they fade more quickly.4. In the case of agranulocytosis mature neutrophils are stained more lightly than those in normal persons.5. In the case of essential hypochromic anemia mature neutrophils in the bone marrow are stained more deeply and earlier than those in normal persons, but fading more slowly. In the case of pernicious anemia these neutrophils are stained more deeply both before and after the treatment than those in normal persons, but as for the fading before and after treatment, the former is slightly faster than the latter.6. These results indicate that, although this method is slightly inferior to the conventional method from the standpoint of neutral red vital staining, it is rather a superior method for clinical application because of its simplicity in manipulation the same as in the study of the carbon-particle phagocytosis.