Supravital Staining Research Articles

Assessment of Nili-Ravi buffalo (<i>Bubalus bubalis</i>) semen by MTT reduction assay

MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tet razolium bromide) assay is commonly used to validate the viability of metabolically active cell s. The study was conducted to examine and validate the MTT test to assess the sperm viability of Nili-Ravi buffalo bulls and compare the efficiency of the te st with the supra-vital staining technique (eosin-nigrosine ) and hypo-osmotic swelling test. Fresh semen sampl es from breeding Nili-Ravi buffalo bulls (n = 20) were collected using an artificial vagina. After assess ing the quality of semen for normal parameters, the MTT assay was carried out in phosphate buffer saline. Resu lts revealed a high significant correlation (r = 0.995) between the viability of sperm and the rate of red uction of MTT. The other proportions of some semen samples showed a weak relationship between the eosinnigrosine method (r = -0.32), hypo-osmotic swelling test (r = -0.12) and motility (r = -0.08). However , the MTT assay was found to be superior to other tests a s it was able to determine those sperm which were more than 90% viable. In conclusion, the MTT assay is a simple, robust test that can be used to select Nili -Ravi buffalo bulls on the basis of sperm quality.

Clinical Diagnosis of Oral Cancer

Oral cavity cancer accounts for approximately 3-4% of all malignancies and is a significant worldwide health problem. The Korea Central Cancer Registry estimates that there will be approximately 1500 new cases of oral cancer in Korea. Oral cancer occurs most commonly in middle-aged and elderly individuals. The majority of oral malignancies occur as squamous cell carcinomas and despite remarkable advances in treatment modalities, the 5-year survival rate has not significantly improved over the past several decades, hovering at about 50% to 60%. The unfavorable 5-year survival rate may be attributable to several factors. First, oral cancer is often diagnosed at a late stage, with late stage 5-year survival rates as low as 22%. Additionally, the development of secondary primary tumors in patients with early stage disease has a major impact on survival. The early detection of oral cancer and premalignant lesions offers the promise to cure chance of oral cancer. The major diagnostics moddalities for oral cancer include oral cavity examination, supravital staining, oral cytology, and optical detection systems. But the clinical finding of oral mucosa is the most important key to confirm the oral cancer until now. The traditional clinical examination of oral cavity can be performed quickly, is without additional diagnostic expense to patients, and may be performed by health care professionals. Therefore, clinicians must be well-acquainted with clinical characteristics of oral cancer and practice routine screening for oral cancer in dental clinic to decrease the morbidity and mortality of disease.

Reticulocytes indices in β thalassemia trait individuals

The thalassemias are a diverse group of microcytic and hemolytic anemias that are characterized by defective synthesis of one of the globin chains of adult hemoglobin. The β-thalassemias are characterized by a reduced (β+-thalassemia) or an absence (β0-thalassemia) of β globin synthesis which leads to imbalanced globin chain production.(1,2) This imbalanced synthesis results in a variable degree of anemia which stimulates erythropoietin production, leading to proliferation and expansion of the bone marrow. The β-thalassemias have a considerable phenotypic variation depending on multiple factors, which include the nature of the mutation involved. This leads to a wide range of presentations from profound anemia (requiring lifelong blood transfusions - β-thalassemia major) to extremely mild anemia (β-thalassemia trait).(2) Reticulocytes are juvenile red blood cells; they contain remnants of the ribosomal ribonucleic acid which is present in large amounts in the cytoplasm of the nucleated precursors from which they are derived. The number of reticulocytes in the peripheral blood is a fairly accurate reflection of erythropoietic activity assuming that reticulocytes are released from the bone marrow after the 'normal' time, and that they remain in circulation for the 'normal' period of time.(3) A total of 152 unrelated adults were included in this study: thirty with the βthalassemia trait diagnosed by high-performance liquid chromatography (HPLC-Variant, Bio-Rad, Milan, Italy)(4) with sequencing of the HBB gene using the primers described by Kimura(5) and Miranda(6) and 122 individuals recruited during their routine blood counts at the Pharmacy School Laboratory of the Universidade Federal do Rio Grande do Sul. The Ethics Committee of Hospital de Clinicas de Porto Alegre, Rio Grande do Sul approved the study protocol. Peripheral blood was collected using EDTA as anticoagulant. Hematological and reticulocyte data were obtained in an automated cell counter - Sysmex SE9500 (Sysmex, Kobe, Japan). Table 1 shows the hematological indices for β-thalassemic trait and control individuals. Table 1 Mean and reference ranges for hematology laboratory values in the Municipal Laboratory of Curitiba, PR Individuals with the β-thalassemic trait presented with significantly higher levels (p-value < 0.05) of the following variables compared to controls: reticulocytes (percentage and number), medium fluorescence reticulocytes, high fluorescence reticulocytes and immature reticulocyte fraction. These results are in agreement with those reported by Noronha & Grotto with the exception of the immature reticulocyte fraction, where no statistical difference was seen between the β-thalassemic trait and control group.(7) In this study no significant difference was found between the groups for low fluorescence reticulocytes. The reticulocyte count is used as an indicator of the erythropoietic activity of bone marrow in different anemias.(7) Manual techniques (such as supravital staining) have great interand intra-observer variability and often the results are inaccurate. Automated cell counting has overcome this limitation. The availability of reticulocyte maturation indices, based on the measurement of RNA content extends the clinical utility of reticulocyte determination.

Manual and automated reticulocyte counts

Manual reticulocyte counts were examined under light microscopy, using the property whereby supravital stain precipitates residual ribosomal RNA versus the automated flow methods, with the suggestion that in the latter there is greater precision and an ability to determine both mature and immature reticulocyte fractions. Three hundred and forty-one venous blood samples of patients were analyzed of whom 224 newborn and the rest adults; 51 males and 66 females, with ages between 0 and 89 years, as part of the laboratory routine for hematological examinations at the Clinical Laboratory of the Hospital Universitário do Oeste do Paraná. This work aimed to compare manual and automated methodologies for reticulocyte countings and evaluate random and systematic errors. The results obtained showed that the difference between the two methods was very small, with an estimated 0·4% systematic error and 3·9% random error. Thus, it has been confirmed that both methods, when well conducted, can reflect precisely the reticulocyte counts for adequate clinical use.

A flow cytometry based in vitro micronucleus assay in TK6 cells—Validation using early stage pharmaceutical development compounds

The micronucleus test (MNT) is a well established test for detecting clastogenic and aneugenic compounds. Despite the assay's advantages, the MNT may produce false positive and false negative results in some conditions. This fact may be related to the underestimation of apoptosis or necrosis, the p53 status of the cell system or the cytotoxicity assay, and the top dose selection. The purpose of our studies was to contribute to the validation efforts of the flow cytometry based MNT. To identify the most reliable cytotoxicity assay for the top dose selection five parameters for relative survival were tested: relative cell count, relative population doubling, trypan blue supravital staining, relative ratio of scored nuclei to latex beads, and ethidium monoazide staining. For all compounds the least sensitive method was the relative cell count and the most reliable was the nuclei/beads ratio. The comparative evaluation of micronuclei induction in TK6 cells, analyzed with microscopy and flow cytometry, was performed with reference compounds and internal Novartis early development compounds with positive, weak positive, equivocal, and negative genotoxic effects. Our data document a good correlation between the MNT results obtained by flow cytometry and by microscopy. The results confirm that the method may be applied for routine testing in the pharmaceutical industry for the tested group of compounds, including compounds which require metabolic activation. However, further validation and miniaturization may be required.

Microwave Ablation of Lung Tissue: Impact of Single-Lung Ventilation on Ablation Size

Thermal ablation is increasingly used to treat pulmonary tumors in medically inoperable patients. Most procedures are performed with sedation in the radiology suite. Ideally, the ablation should encompass the entire tumor volume with a surrounding margin of necrosis; however, ablation may not be as effective in the normal aerated lung surrounding a denser tumor. Inducing atelectasis of the lung may potentially increase ablation volumes and increase local cancer control. This study examines the effect of single-lung ventilation on ablation size using a microwave system. Twenty microwave ablation procedures were performed in the lungs of 10 swine. Bilateral thoracotomy using a clamshell approach was used. In one lung, ablation was performed with continuous ventilation. In the contralateral lung, single-lung ventilation was achieved by clamping the bronchus before ablation. The ablated lobes were resected and sent for pathologic analysis. Routine and supravital staining was performed. The ablation zone was clearly demarcated on gross examination, and in all cases 100% ablation occurred, without skip areas of viability. The ablation zones were elliptical with the long axis parallel to the axis of the ablation probes (active tip, 3.7 cm). Ablation diameters and volume were compared between the ventilated and nonventilated lungs. Ablation volume was superior in nonventilated lungs (10.74 cm(3) versus 7.35 cm(3); p = 0.039) primarily because of differences in the short axis of the ablation zone. Microwave energy can effectively ablate normal pulmonary parenchyma without skip areas of viable tissue within the gross ablation field. The volume of necrosis is increased in nonventilated lungs, suggesting that ablation results can be improved in patients by using general anesthesia with single-lung ventilation. Future studies will be required to confirm this hypothesis.

The Diagnosis of α-Thalassaemia: A Case of Hemoglobin H-α Deletion.

We report a case of hemolytic anemia that was subsequently identified to be a case of α-thalassaemia harboring the common rightward 3.7kb deletion/HbH. The diagnosis was based on sequential analyses using BioRad D10 HPLC, Alkaline gel electrophoresis, GPO α THAL-IC strips and the identification of the specific genetic lesion using an α Globin reverse dot blot hybridization assay. Supravital stain of RBCs helped in identifying classical HbH inclusions. In a background of a variable clinical presentation, lack of definitive hematological markers, and general under-diagnosis of α-thalassaemias we have used this case to highlight the features and sequence of techniques involved in identifying and characterizing an α-globin chain mutation, starting from a diffuse clinical history and presentation up to the identification of a specific genetic lesion involved.

Influence of sperm concentration on the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm during liquid storage

The aim of this study was to determine the influences of two different concentrations in terms of motility, morphology, membrane integrity (viability and HOST response: HE-test; modified hypoosmotic swelling test (HOST) associated with supravital eosin staining test), DNA integrity (COMET assay) and oxidative stress parameters (MDA, malondialdehyde; AOA, total antioxidant activity; GSH, reduced glutathione; NOx, nitric oxide) of liquid stored ram sperm for 5 days. Two different concentrations suitable for laparoscopic and cervical inseminations were employed. A total of 5 Pirlak rams (Daglic × Kivircik, local breed) with satisfactory breeding potential were selected. Semen samples were collected by artificial vagina. Ejaculates were extended to 25 and 100 million sperm per ml with Tris-based egg-yolk (T) extender at room temperature and stored at 4 °C. The concentration significantly had effects on motility, morphology, membrane and DNA integrity along with oxidative stress parameters ( P < 0.05). Overall changes in the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm diluted with T in the 100 × 10 6 ml −1 concentration and preserved at 4 °C for a short term was found to be better preservation than that of diluted with T in the 25 × 10 6 ml −1 concentration. Significant concentration × days of storage interactions were found for all parameters except the NOx. Further studies are required to elucidate the oxidative stress changes on sperm quality parameters in different concentrations during the liquid storage of ram semen.

Effect of seminal plasma on functional integrity of rabbit sperm membranes during storage at 4ºC or freezing.

The effect of semen plasma removal either by simple centrifugation or by separation through a Percoll gradient on the integrity of plasma membranes of rabbit spermatozoa during storage at 4ºC and freezing was evaluated in two successive experiments. A modifi ed hypo-osmotic swelling test procedure combined with supravital staining was employed to evaluate simultaneously membrane integrity of head and tail membranes of sperm cells. In the fi rst experiment, the impact of semen plasma on membrane integrity of sperm cells was examined in Tris-citric acid-glucose extender at 4ºC for 96 h. The percentage of sperm cells with disintegrated tail and head membranes increased in all groups in correlation with the length of storage. After storage for 96 h, removal of semen plasma, irrespective of the method of removal, resulted in signifi cant increase (P&lt;0.01) in the percentage of sperm cells with disintegrated plasma membranes. The adverse effect of storage and removal of semen plasma was more prominent on the tail membranes, and especially during the fi rst 24 h of the storage period. In the second experiment, the impact of semen plasma on membrane integrity of sperm cells undergoing freezing was examined. A total of three groups were arranged as described in the fi rst experiment, and semen samples were frozen in straws using an extender including acetamide and methyl cellulose. Freezing of semen drastically reduced the percentage of sperm cells with intact plasma membranes in postthaw samples. However, removal of semen plasma, irrespective of the method of removal, did not affect the proportion of sperm cells with intact plasma membranes. In conclusion, the effect of semen plasma on plasma membranes varied signifi cantly relative to the preservation temperature of sperm cells. Although it exerted a protective infl uence during storage at 4ºC, no protective impact was monitored during freezing.

Effect of bendiocarb on development of the chick embryo

Agrochemicals, including pesticides, are being used in increasing amounts in agriculture and are therefore potential environmental contaminants which may affect a variety of biological systems. The pesticide residues directly affect the embryos, disturbing their normal development and causing pathophysiological and morphological changes. We have observed embryotoxicity of cholinesterase inhibitor bendiocarb in the chick embryo. The pesticide dissolved in 10% acetone in water for injection was applied in a volume of 200 microl over the embryo through membrana papyracea at embryonic days (ED) 2, 3, 4, 5 and 10. The toxicity of bendiocarb was rather low, and lethal dose (LD(50)) decreased with advancing development from 0.97 mg per egg at ED 2 to 28.6 mg on ED 5. The malformations in surviving embryos were observed rarely (<2%) and occurred in both control and experimental groups. In the treatment at ED 5 and 10 there was a statistically significant reduction in body weight, but the maximum difference from controls was below 14%. In treated chick embryos on ED 3 and 4 was observed a small but not significant increase in number of dead cells using supravital staining. We conclude that bendiocarb possesses no significant toxicity in the avian embryo. Our analysis of bendiocarb embryotoxic potential in the chick embryo supports the earlier observations in other animal models, testifying to the relative safety of bendiocarb for the embryo or fetus.

Potent spermicidal effect of oleanolic acid 3-beta- d-glucuronide, an active principle isolated from the plant Sesbania sesban Merrill

Background The spermicidal activity of oleanolic acid 3-β- d-glucuronide (OAG), an active principle isolated from root extracts of Sesbania sesban, was evaluated. Study Design Under the Sander–Cramer test criteria, the sperm-immobilizing activity of OAG was studied using highly motile rat sperm. Sperm mortality and membrane integrity were assessed by supravital staining, hypo-osmotic swelling (HOS), transmission electron microscopy (TEM) and sperm membrane lipid peroxidation (LPO). In vitro microbicidal potential and hemolytic index of OAG were examined in Lactobacillus culture and rat red blood corpuscles (RBCs), respectively. Post-intravaginal OAG application, the in vivo contraceptive efficacy was evaluated in rats. Ames test determined the carcinogenic potential of OAG. Results The minimum effective concentration (MEC) of OAG was 50 mcg/mL. More than 97% of the OAG-treated sperm lost their HOS responsiveness in a dose-dependent manner. TEM and LPO revealed that OAG affected the sperm membrane integrity. OAG declined fertility to zero, was nonmutagenic and was not harmful to lactobacillus. Conclusion OAG has significant spermicidal activity that may be explored further.

Cholesterol-loaded cyclodextrin enhances osmotic tolerance and inhibits the acrosome reaction in rabbit spermatozoa

The effects of cholesterol-loaded cyclodextrin (CLC) treatment on the osmotic tolerance and ability to undergo the acrosome reaction of rabbit spermatozoa, with an unusually high cholesterol/phospholipid ratio in plasma membranes, were examined in two successive experiments. In the first experiment, CLC-pretreated and untreated sperm cells were exposed for 15 min to one of five fructose solutions, adjusted to 20, 80, 290, 500 or 1500 mOsm/L. After the anisoosmotic challenge, the integrity of sperm membranes in the CLC-supplemented (at a dose level of 3 mg/120 × 10 6 spermatozoa) and control groups was estimated by a modified hypoosmotic swelling test (HOST) associated with a supravital eosin staining test (HE-test). In the second part of the study, the influence of cholesterol supplementation on the acrosome reaction of sperm cells stimulated by either calcium ionophore A23187 (CI) or lysophosphatidylcholine (LPC) was evaluated. CLC pretreatment increased viable and live-HOST-responsive sperm rates ( P < 0.01) after incubation in anisoosmotic solutions varying from 80 to 1500 mOsm/L. However, CLC supplementation did not influence the percentage of HOST-responsive sperm cells ( P > 0.05). A significant interaction was determined between CLC pretreatment and the level of osmotic pressure in maintaining the functional and physical integrities of sperm membranes undergoing osmotic challenges. Both CI and LPC successfully induced the acrosome reaction in rabbit spermatozoa ( P < 0.001). Compared with CI, LPC was more effective ( P < 0.0001). CLC pretreatment resulted in a significant reduction ( P < 0.01) in the percentage of acrosome reacted sperm cells irrespective of the inducing agent, either CI or LPC. In conclusion, CLC treatment enhanced the anisoosmotic tolerance of rabbit spermatozoa and reduced their ability to undergo the acrosome reaction after stimulation by CI or LPC.

Assessment of buffalo semen with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which measures the reduction of MTT, is commonly used to validate the viability of metabolically active cells. This study was conducted to evaluate and validate the MTT assay to assess the spermatozoal viability of Nili-Ravi buffalo bulls and compare the efficiency of the test with the supravital staining technique (eosin and nigrosin) and the hypoosmotic swelling test. Fresh semen samples from breeding Nili-Ravi buffalo bulls (n = 25) were collected using an artificial vagina. After assessing the quality of the semen for routine variables, the MTT assay was carried out in PBS. Results revealed a correlation (r = 0.979; P < 0.001) between the viability of spermatozoa and the rate of reduction of MTT. The other proportions of same semen samples showed a poor relationship between the eosin and nigrosin test (r = -0.25), the hypoosmotic swelling test (r = -0.12), and motility (r = -0.15). However, the MTT assay was found to be superior compared with other tests because it was able to determine those spermatozoa that were more than 90% viable. In conclusion, the MTT assay is a simple, robust test that can be used to select Nili-Ravi buffalo bulls on the basis of spermatozoa quality.

11 FERTILITY EVALUATION OF THE GOAT SEMEN DILUTED IN CITRATE-EGG YOLK EXTENDER WITH HIGH OR LOW EGG YOLK CONCENTRATION, COOLED AND STORED AT 5°C FOR 24 HOURS

This experiment was designed to evaluate, in vitro and in vivo, the cooling resistance of the goat semen diluted in citrate-egg yolk extender with high (20%) or low (2.5%) concentration of egg yolk. The experiment was conducted at Dairy Goat Station of the Animal Science Department (Federal University of Viçosa, MG, Brazil), during the natural breeding season (March to June). Four bucks of the Alpine (n = 2) and Saanen (n = 2) breeds were used as semen donors. The bucks did not have apparent abnormalities of the reproductive tract and the fertility was proved based on andrologic examination and the records from the previous breeding season. The semen was collected by the artificial vagina method and a fresh sample was analyzed for the sperm cell motility and concentration. Therefore, the semen was diluted in citrate-egg yolk extender with high (T1 = 20%) or low (T2 = 2.5%) concentration of egg yolk, cooled and stored at 5°C for 24 h, before being used either for quality analysis or for AI. Reproductive-age female Saanen and Alpina goats (n = 68) were inseminated via cervix with cooled semen from T1 (n = 32) or from T2 (n = 36). The parametric data from cooled semen quality (sperm cell motility, vigor, hyposmotic swelling test, morphology, and supravital staining) were analyzed by ANOVA, for the main effect of the treatment, using the SAEG 9.0 program (UFV, 2005). The differences between means were accessed by the Student-Newman-Keuls test. The qualitative data from pregnancy rate were tested by chi-square (Ayres et al. BioEstat 2.0, 2000). The mean ± SEM for each quality test of the cooled semen, as well the P-value for comparisons between treatments were as follows: motility, 68.6 ± 15.5 and 78.0 ± 5.6% (P &lt; 0.05); vigor, 2.5 ± 0.6 and 3.2 ± 0.3 (P &lt; 0.05); supravital staining, 59.4 ± 17.3 and 71.2 ± 10.6% (P &lt; 0.05); and hyposmotic test, 42.3 ± 15.8 and 56.3 ± 11.6% (P &lt; 0.05), respectively, for T1 and T2. The pregnancy rate was higher (P &lt; 0.05) in goats inseminated with cooled semen from T2 (66.7%) than in those inseminated with T1 (31.25%). The number of the offspring did not differ (P &gt; 0.05) between treatments (1.3 ± 0.5 and 1.4 ± 0.5 kids, forT1 andT2, respectively). The interpretation was that the goat semen diluted, cooled, and stored in citrate-egg yolk extender prepared with a low (2.5%) concentration of egg yolk produced higher pregnancy rate and the tests of semen quality were sensitive to assess this difference. Financial support was provided by CnPQ (Brazil).

Evaluation of spermicidal potential of labdane diterpenes from Andrographis paniculata

Andrographolide, 14-deoxyandrographolide, andrograpanin, andrographiside, deoxyandrographiside, and neoandrographolide were isolated from the leaves of A. paniculata and evaluated for their spermimmobilizing activity. The mode of spermicidal action was assessed by (a) supravital and double fluoroprobe staining of sperm, (b) hypo-osmotic swelling test, and (c) transmission electron microscopy. Contraceptive efficacy was evaluated by intrauterine application in rat followed by mating, and subsequent assessment of the pregnancy outcome. 14-Deoxyandrographolide with MEC of 200 mg/ml was found to be the most active among the six compounds. It is comparable in activity to nonoxynol (N-9) and exerts its effect in a dose dependent manner. Intra-vaginal administration of 10% 14-deoxyandrographolide did not cause any significant vaginal irritation in rat. The findings strongly suggest that 14-deoxyandrographolide may be used as a nondetergenttype spermicidal agent.Key words: sperm-immobilizing activity; andrographolide; 14-deoxyandrographolide.

The best stain for morphological study of human seminal fluid's smears.

Objectives:There is a high need for proper evaluation of the morphological features of human sperms. The importance of this lies in the field of andrology, male fertility and in vitro fertilization. The wet smears can give rough clue about the shape of the sperms, but it is neither accurate nor reproducible. This study aimed to determine the best stain which can be used for seminal fluid cytology.Methods: This study was conducted in Port Sudan, Red Sea State, Sudan in the period from October 2006 to September 2007. The total number of patients was 50. Samples which were collected from normospermic patients (NSP) were prepared by direct smear technique. Samples which were collected from oligospermic patients (OSP) and azoospermic patients (ASP) were prepared by direct smear technique and also by indirect smear techniques (concentration method). Smear samples were stained by freshly prepared Harris's Haematoxylin, Papanicolaou stain, May-Grunwald Giemsa stains (MGG), supra vital stain, Giemsa stain and leishman's stain.Results: In this study, the best stain was Harris's Haematoxylin (80% excellent for the head of sperm, 70% good for the neck, 59% excellent for the tail, 42% very good for cells in background). Harris's stainwas followed by papanicolaou stain and the third best stain was supra vital stain. MGG was better than Giemsa in staining of semen smears (75% good versus 25% good) in overall performance. The worst stain was Leishman's stain.Conclusion: Stained smears must be used for the morphological study of semen samples. Harris's Haematoxylin is the best stain for semen cytological features. Stains which used for the semen samples should befreshly prepared.

Determination of red cells, nucleic acid‐containing cells and platelets (RNP Determination) by a crossover analysis of emission DNA/RNA light

We developed a new flow cytometry analysis method using a crossover analysis of emission light from intracellular DNA/RNA. Both the RNA and the DNA content in each cell were new parameters obtained by fluorescence analysis using acridine orange supravital stain. With this method, two-dimensional diagrams produced by cellular RNA concentration (CRc) and cellular DNA concentration (CDc) enabled clear separation of red cells and platelets, and the diagram (RNP Diagram) also distinguished fluorescently stained blood cells and small particles derived from background dust of the reagent. This study assessed the capability of RNP Determination concerning reticulocyte count and accurate platelet count obtained by the ratio of red cells and platelets. The distance of each event between red cell distribution and platelet distribution was sufficiently large on the diagram, and the two regions did not overlap. Both reticulocyte count and platelet count showed excellent correlation with those obtained by their respective reference methods. In conclusion, this new assay, using RNP Determination, demonstrated great potential for detecting abnormalities of red cells and platelets.

10th Annual Congress of the German Association of Clinical Pharmacology

10th Annual Congress of the German Association of Clinical Pharmacology

Neutral red supravital staining for cellular elements in the semen.

Human seminal fluid besides spermatozoa often contains other cellular elements. A supravital staining method designed to differentiate the above mentioned cellular elements was described. Amongst the spermatogenic cells only spermatocytes were stained with Neutral Red. They displayed two peculiar structures designated as "Y" granules and "Enigmatic Body". Neutral Red was absorbed by the spermiophage cells and was concentrated by them in the form of cytoplasmic granules. In addition the coarse granules of leukocytes and the gigestive vacuoles of Balantidium Coli and Trichomonads were stained with Neutral Red.

Fluorescence Supravital Stain of Human Sperm: Correlation with Sperm Motility Measured by a Transmembrane Migration Method/Supravital-Fluoreszenz-Färbung von menschlichen Spermatozoen: Korrelation zur Spermatozoenmotilität (Messung mittels Transmembran-Mi

We developed a supravital stain of human sperm with fluorescent dyes. Either Hoechst 33,258 or fluorescein isothiocyanate could be used, the former stained sperm head while the later stained the whole sperm. Sperm vitality assessed with any of these two fluorescent dyes correlated well with that determined by eosin-nigrosin counterstain. When sperm vitality was compared with sperm motility measured with a transmembrane migration method, we found that many vital sperm were immotile because sperm vitality was higher than sperm motility in tested samples.