A point of confusion for embryologists in the identification of viable spermatozoa by the eosin-nigrosin test

Chen, Huanhua,Zhou, Hong,Shu, Jinhui,Gan, Xianyou,Wang, Caizhu,Lin, Ruoyun The Korean Society for Reproductive Medicine 2019 Clinical and Experimental Reproductive Medicine Vol.46 No.1

A viable spermatozoon is a prerequisite for fertilization in intracytoplasmic sperm injection (ICSI). Thus, it is crucial to select viable but immotile spermatozoa on the day of ICSI. We report conflicting results in the identification of viable but immotile spermatozoa between the eosin-nigrosin staining and the laser test, which resulted in confusion for embryologists during assisted reproductive technology (ART). Three patients' semen samples that showed no motile spermatozoa are described in this report. To identify viable spermatozoa, we used both the eosin-nigrosin test and the laser test for each sample, and repeated the semen analysis twice in each patient. Viable but immotile spermatozoa selected by the laser test were used for ICSI. Viable spermatozoa were detected by both the eosin-nigrosin and laser tests in two patients (case 1, 95.00% vs. 24.21% and 92.68% vs. 22.22%; case 2, 41.18% vs. 23.48% and 39.81% vs. 22.52%), indicating consistent results between the two methods. In the third patient, the eosin-nigrosin test yielded viability rates of 20.75% and 19.14%, while the result of the laser test was 0%. Thus, testicular aspiration was performed to collect viable sperm from this patient. Normal fertilization was achieved after the injection of viable but immotile spermatozoa selected from these patients by the laser test, resulting in the birth of two healthy babies. Our study documents a case where the eosin-nigrosin test showed a limitation in identifying viable but immotile spermatozoa for ART, while the laser test may overcome this limitation. Larger samples may be required to corroborate the clinical value of the laser test.

배경에 따른 혈문증강 시약(Fuchsin Acid, Eosin-Y, Acid Yellow 7) 적용의 현출도 비교실험

김아람(A-Ram Kim),김우중(Woo-Joong Kim),정혜영(Hey-Young Jung) 한국콘텐츠학회 2013 한국콘텐츠학회논문지 Vol.13 No.6

강력사건에서 혈액지문은 범인과 피해자를 연결하는 중요한 증거물이기에 현재까지도 이에 대한 많은 실험과 연구가 이루어지고 있다. 과거부터 Fuchsin Acid와 Acid Yellow7간의 비교는 이루어져왔으나, Eosin-Y의 경우 국내에 조직염색시약으로만 알려졌을 뿐 직접적으로 지문에 적용된 사례가 없다. Fuchsin Acid는 특유의 붉은색으로 인해 밝은 배경에서 좋은 대조비를 보이지만, 어두운 배경에서는 Gel lifter를 적용해야 형광이 관찰되는 등 번거로움이 있다. 또한 Gel lifter 사용 시 지문의 좌우가 바뀌기 때문에 주의를 기울여야 한다. Acid Yellow 7은 노란색의 결과물이 현출되기 때문에 밝은 배경에서는 대조비가 좋지 않지만, 어두운 배경에서 매우 강한 형광을 나타냈다. Eosin-Y의 경우 분홍색의 결과물로 인해 밝은 배경에서 Acid Yellow 7에 비해 대조비가 좋았으며, 어두운 배경에서는 Fuchsin Acid 보다나은 형광이 관찰되었다. 물론 Eosin-Y가 밝은 배경에서의 Fuchsin Acid 및 어두운 배경에서의 Acid Yellow 7 형광에 비해서 효과성은 다소 떨어지나 배경에 구애받지 않는다는 장점이 있기에 현장에서 근무하는 수사요원에게는 보다 효과적인 혈액지문증강이 될 것이다. In serious crimes, bloody fingerprints are crucial evidence that make links between suspects and victims. There have been many studies related to bloody fingerprints for a long time. There are many comparative studies for effectiveness of Acid Fuchsin and Acid Yellow 7, but nothing about Eosin-Y in this country. Acid Fuchsin is a useful reagent that has unique red color distinguishing from light colored background. but it is useless on dark surfaces. In order to make it visible, we should use BVDA Gel lifters. On the contrary this, Acid Yellow 7 makes stronger fluorescence on a dark background. In this study, we got the conclusion that Eosin-Y is more useful than the others not only on dark background but also light background.

무정자증 환자의 고환 정자내 Chromatin Condensation 검사를 위한 Aniline Blue-eosin 염색법의 유용성

박용석,손지흔,이중식,서주태 대한남성과학회 2010 The World Journal of Men's Health Vol.28 No.2

Purpose: This study was performed to evaluate chromatin condensation of morphologically mature sperm using a modified aniline blue-eosin (AB-E) staining method in azoospermia. Materials and Methods: Chromatin condensation was analyzed using an AB-E staining method in 61 cases (50 patients) of TESE or testicular biopsy with the patient’s own sperm. Obstructive azoospermia (OA) was present in 48 cases in 39 patients and non-obstructive azoospermia (NOA) was present in 13 cases in 11 patients, respectively. Immature sperm heads were stained dark blue, whereas mature sperm were stained red-pink by the eosin. The percentage of sperm chromatin condensation was calculated from the ratio of the number of red-pink sperm to the total number of sperm analyzed. Results: The percentage of chromatin maturity was 37.7% vs. 30.3% in OA and NOA, respectively, of the total sperm cell count. The maturity of fresh testicular sperm was 38.3% and 36.3% in OA and NOA, respectively. Also, the maturity of thawed testicular sperm was 34.5% and 10.3% (p<0.05) in OA and NOA, respectively. The maturity of fresh and thawed testicular sperm was 36.3% and 10.3% (p<0.05), respectively, in NOA. These results suggest that chromatin condensation is less stable in sperm of NOA and freezing and thawing procedures may impair sperm chromatin condensation. Conclusions: In our results, the aniline blue-eosin staining method improved the visualization of excessive histones in sperm and the diagnosis of sperm immaturity in morphologically normal testicular sperm. We found that AB-E staining method can be an effective method for analyzing testicular sperm chromatin condensation in azoospermia.

Analysis of S-glutathionylated proteins during adipocyte differentiation using eosin-glutathione and glutaredoxin 1

( Sungwon Hwang ),( Sana Iram ),( Juno Jin Inho Choi ),( Jihoe Kim ) 생화학분자생물학회 2022 BMB Reports Vol.55 No.3

Protein S-glutathionylation is a reversible post-translational modification on cysteine residues forming a mixed disulfide with glutathione. S-glutathionylation, not only protects proteins from oxidation but also regulates the functions of proteins involved in various cellular signaling pathways. In this study, we developed a method for the detection of S-glutathionylated proteins (ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin 1 (mGrx1). ProSSG was efficiently and specifically labeled with E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection of ProSSG with semi-quantitative determination. In addition, a deglutathionylation enzyme mGrx1 specifically released E-GSH from ProSSG-E, which increased fluorescence allowing a sensitive determination of ProSSG levels. Application of the method to the adipocyte differentiation of 3T3-L1 cells showed specific detection of ProSSG and its increase upon differentiation induction, which was consistent with the result obtained by conventional immunoblot analysis, but with greater specificity and sensitivity. [BMB Reports 2022; 55(3): 154-159]

Evaluation of Boar Sperm Viability by MTT Reduction Assay in Beltsville Thawing Solution Extender

Byuna, J.W.,Choo, S.H.,Kim, H.H.,Kim, Y.J.,Hwang, Y.J.,Kim, D.Y. Asian Australasian Association of Animal Productio 2008 Animal Bioscience Vol.21 No.4

MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) reduction assay is a method that validates the viability of an active cell. Dehydrogenase in mitochondria converts yellow colored insoluble tetrazolium salt to purple colored water-soluble formazan. Sperm also have mitochondria in the midpiece, therefore sperm viability could be evaluated by MTT reduction assay. Several studies have already demonstrated the capability of application of the MTT reduction assay to sperm of several species in Hepes-BSA buffer. Because most liquid semen was diluted in extender like BTS (Beltsville Thawing Solution), Modena or Androhep when it is used or transferred, semen needed another dilution in Hepes-BSA buffer to assess sperm viability. In this study, we evaluated boar sperm viability especially in BTS extended semen and compared the efficiency of this test with eosin-nigrosin staining. We used the fresh BTS extended semen from a local A.I center. Semen sample was diluted to $3.0{\times}10^7$ sperms/ml in BTS. The rates of formazan production were measured in 96-well microtiter plates immediately and 1h after incubation at $17^{\circ}C$ using a spectrophotometer at wave length 560 nm. Simultaneously, split samples of the same semen were tested, using eosin-nigrosin staining to compare the efficiency of the MTT assay of sperm viability in BTS. The correlation between the results of these tests was calculated using Student-t test and ANOVA. The results revealed a strong correlation between the results of MTT reduction rate and the results that were simultaneously determined by eosin-nigrosin staining at 1 h. In conclusion, the MTT reduction test was an effective and simple method to validate sperm viability and it could be used as a simple tool to evaluate sperm viability in the local A.I center and laboratory.

Fabrication of CdS and CuWO4 modified TiO2 nanoparticles and its photocatalytic activity under visible light irradiation

K. Vignesh,A. Suganthi,M. Rajarajan,R. Priyanka,R. Hariharan 한국공업화학회 2014 Journal of Industrial and Engineering Chemistry Vol.20 No.2

CdS and CuWO4 modified TiO2 nanoparticles (CdS–CuWO4-TiO2) were prepared by the chemicalimpregnation method. The as-prepared nanoparticles were characterized using UV–visible-diffusereflectance spectroscopy (UV–vis-DRS), powder X-ray diffraction (XRD), scanning electron microscopy(SEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS) and B.E.T. surface area analysis techniques. The photocatalytic activity was evaluated based on the degradation of adye (eosin-Y) and inactivation of a bacterium (Pseudomonas aeruginosa). The results revealed that CdS–CuWO4-TiO2 showed high photocatalytic activity over CdS-TiO2, CuWO4-TiO2 and TiO2. Moreover thereusability and stability of the photocatalyst for the degradation of eosin-Y was also studied.

A comparative study of efficacy of coconut oil, lemon water and dishwashing liquid as surrogates to xylene

Ayeesha Thajudeen,Sudha Srinivasan,GeethaPriya Govindarajan,Akashavanan Shanmu 환경독성보건학회 2022 환경독성보건학회지 Vol.37 No.3

Histopathology is the field of science that helps in analyzing the architectural pattern of cells under the microscope. Hematoxylin and eosin-stained sections are used for routine histopathological examination. Xylene being a biohazardous hydrocarbon is used in many steps of tissue processing and laboratory personnel are exposed to this toxic substance. Maximum exposure to xylene occurs in the step of deparaffinization, for which alternate safer methods should be introduced. This study compares the efficacy of natural products like coconut oil, lemon water, less chemical substance like dish wash liquid with xylene as deparaffinizing agent. 50 paraffin embedded sections were used in each of the groups using xylene, coconut oil, diluted lemon water and dish wash liquid as deparaffinizing agents. 80% of slides using dishwashing liquid, 64% using lemon water and 42% of slides using coconut oil showed excellent cellular features. 96% of slides using xylene showed good quality staining, 54% of slides using dishwashing liquid and 40% slides using lemon water showed good quality staining. Only 4% of slides prepared using coconut oil showed good quality staining. Dishwashing liquid is the best surrogate and among the natural products, diluted lime water yields a better result and coconut oil, the least productive as deparaffinizing agent in this study.

고환조직 동결-융해 후 회수된 고환 정자에 대한 Hypo-osmotic Swelling (HOS) Test의 효과

박용석,이형송,송상진,김정욱,강인수,서주태,Park, Yong-Seog,Lee, Hyoung-Song,Song, Sang-Jin,Kim, Jeong-Wook,Kang, Inn-Soo,Seo, Ju-Tae 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.3

Objectives: We have previous reported that thawed testicular sperm and sperm extracted from seminiferous tubule could achieved optimal fertilization and pregnancy in azoospermic patients. However, thawed testicular sperm did not show motility in many cases. Therefore we studied viability of immotile sperm extracted from frozen-thawed seminiferous tubule using hypo-osmotic swelling (HOS) test and eosin-Y test. Materials and Methods: After sperm extraction using for ICSI, the remained sections of seminiferous tubules were frozen with a computerized freezer. For thawing and preparation of testicular sperm, the seminiferous tubules were thawed by removing from $LN_2$ and letting them at room temperature for 10 min followed by %37^{\circ}C$ water bath for 10 min. The prepared samples were washed for free of preservation medium and sperm preparation method described previous. Sperm was suspended in 0.1 ml hypoosmotic solution. After 30 minutes, the type of distally coiled sperm were assessed. Results: In 44 cases of cryopreservation of seminiferous tubules in obstructive azoospennic patients, the fertilization rates with 2PN were 71.4% and pregnancy rates were 34.1%. The presence of motile spermatozoa on subsequent post-thaw testicular sperm remarked 15.1% and were increased to 77.3% just before ICSI. After sperm extracted from frozen-thawed seminiferous tubule, 3 hrs later in in vitro culture, the cases of presence of motile sperm, reaction of hypo-osmotic swelling test and viable sperm were 63.6% (28/44), 93.2% (41/44), and 77.3% (34/44), respectively. Conclusions: Just after post-thawed testicular sperm did not showed motility. Although motility was gained after in vitro culture, many cases showed non-motile sperm until optimal insemination time. However, HOS test showed positive reaction in non-motile sperm. Therefore, HOS test is an alternative method for the selection of viable sperm for ICSI.

입자상 활성탄에 의한 Eosin Y의 흡착제거

이종집(Jong-Jib Lee) 한국청정기술학회 2010 청정기술 Vol.16 No.4

폐감귤박으로 만든 활성탄을 이용한 염료 Eosin Y 흡착에서 반응표면 모델링

감상규 ( Sang-kyu Kam ),이민규 ( Min-gyu Lee ) 한국공업화학회 2018 공업화학 Vol.29 No.3

반응 표면법(RSM)과 Box-Behnken 설계(BBD) 통계 방법을 사용하여 폐감귤박으로 만든 활성탄(WCAC)에 의한 염료Eosin Y의 흡착을 검토하였다. 실험은 Eosin Y의 농도(Conc. : 30~50 mg/L), 용액 온도(Temp. : 293~313 K) 및 흡착제투여량(Dose : 0.05~0.150 g/L)의 3가지 입력 변수를 가진 BBD에 따라 수행하였다. 염료 Eosin Y 제거에 대해 얻어진 2차 다항식 모델의 회귀분석 결정계수(R²) 값이 0.9851이고 적합성 결여(Lack of fit)의 p 값은 0.342로 실험 데이터는 2차 다항식 모델에 잘 부합하였다. 염료 농도 50 mg/L, 온도 333 K 및 흡착제 투여량 0.1056 g에서 최적 염료 흡착량59.3 mg/g이 얻어졌다. WCAC에 의한 Eosin Y의 흡착공정은 유사 2차 속도식에 의해 잘 기술되었으며, 등온 실험결과는 Langmuir 모델식을 따랐다. The adsorption of Eosin Y by the activated carbon (WCAC) prepared from waste citrus peel was investigated by using response surface methodology (RSM) and Box-Behnken design (BBD) statistical procedures. Experiments were carried out as per BBD with three input parameters, the Eosin Y concentration (Conc. : 30~50 mg/L), the solution temperature (Temp. : 293~313 K), and the adsorbent dose (Dose : 0.05~0.15 g/L). Regression analysis showed a good fit of the experimental data to the second-order polynomial model with coefficients of the determination (R²) value of 0.9851 and P-value (Lack of fit) of 0.342. An optimum dye uptake of 59.3 mg/g was achieved at the dye concentration of 50 mg/L, the temperature of 333 K, and the adsorbent dose of 0.1056 g. The adsorption process of Eosin Y by WCAC can be well described by the pseudo second order kinetic model. The experimental data followed the Langmuir isotherm model.