아가로스 겔 전기영동에서 간단하고 실용적인 DNA 염색 방법

박성환(Seong-Whan Park) 한국산업기술융합학회(구. 산업기술교육훈련학회) 2022 산업기술연구논문지 (JITR) Vol.27 No.4

핵산 염색시약은 아가로스 겔 전기영동에서 DNA 밴드를 관찰하기 위해 필요하다. 전통적인 염색법은 겔 전기영동이 끝난 후 염색하는 post-staining 방법이지만, 편리한 대안으로 겔에 염색시약을 첨가하는 pre-cast 염색법이 사용되어 왔다. 또 다른 염색 방법으로 염색시약을 아가로스 겔이 아닌 6X 로딩 버퍼에 첨가하는 pre-load 염색법이 있다. Pre-load 염색법은 가장 간단하고 시간을 절약할 수 있는 방법이지만, 동일한 겔 내에서 DNA 밴드의 이동성이 차이를 보일 가능성이 있다. 본 연구에서는 염색시약이 혼합된 Midori Green Direct, GRGreen DNA loading buffer, ENVISION DNA dye loading buffer 등 상용의 loading buffer를 사용할 때, DNA 이동성에 변화가 나타나는 것을확인하였다. 반면에 250X GelRed®를 함유한 자체제작 6X 로딩 버퍼는 다양한 실험 조건에서도 이동성의 변화를나타내지 않았다. 마찬가지로, Shinystar와 EcodyeTM가 포함된 6X 로딩 버퍼 또한 안정적인 이동성을 보여주었다. 결론적으로, pre-load 염색법은 일상적인 겔 전기영동 작업에서 편리하고 실용적인 염색방법이 될 수 있으며, 250X GelRed®를 함유한 자체제작 6X 로딩 버퍼는 pre-load 염색에 있어 유용한 해결책이 될 수 있다. Nucleic acid staining dyes are necessary for the visualization of DNA bands in agarose gel electrophoresis. The conventional staining method follows a post-staining protocol that follows gel electrophoresis; pre-cast staining is used as a convenient alternative. Pre-load staining is another method in which a staining dye is added to the 6X loading buffer, and not in the agarose gel. Pre-load staining is simple and time-saving; however, it could alter the mobility of DNA bands within the same gel. In this study, the mobility alteration owing to the use of commercial pre-mixed loading buffers, such as Midori Green Direct, GRGreen DNA loading buffer, and ENVISION DNA dye loading buffer, were evaluated. Lab-prepared 6X loading buffer containing 250X GelRed®dye did not alter the mobility under various experimental conditions. Similarly, 6X loading buffer containing Shinystar and EcodyeTMdid not affect mobility. In conclusion, pre-load staining method is a convenient and practical staining method for routine gel electrophoresis; lab-made 6X loading buffer containing 250X GelRed®could be used for pre-load staining.

Changes in color and staining of dental composite resins after wear simulation

Lee, Yong-Keun,Lu, Huan,Oguri, Makoto,Powers, John M. Wiley Subscription Services, Inc., A Wiley Company 2007 Journal of Biomedical Materials Research Part B Vol. No.

<P>Objectives: The objectives were to measure the discoloration as well as the change in staining of composite resins after wear simulation. Methods: Generalized wear simulation was performed with a three-body wear testing device for 400,000 cycles for six composite resins. A flat-planed stylus made with polyacetal resin was loaded perpendicularly under a load of 76–80 N. Color of nonworn and worn areas was measured with a spectrophotometer before and after staining with 0.5% methylene blue solution. Nonworn surface served as a control. Differences in color between nonworn and worn surfaces were calculated to indicate the change in color due to wear. Color change after staining with 0.5% methylene blue solution for nonworn and worn surfaces was calculated to observe the changes in staining. Results: Color difference (ΔE<EM><SUP>*</SUP><SUB>ab</SUB></EM>) between nonworn and worn surfaces was in the range of 0.8–1.4 before staining, which increased to 1.1–3.9 after staining. Color change by staining with methylene blue in nonworn surface was 6.8–20.6, and that in worn surface was 5.2–17.8. Therefore, staining in nonworn surface was higher than that in worn surface (p < 0.05). Generalized wear simulation for ∼3 years of clinical service resulted in acceptable color change before staining (ΔE<EM><SUP>*</SUP><SUB>ab</SUB></EM> < 3.3). After staining, color difference between nonworn and worn surface increased to not-acceptable value in one composite resin investigated. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007</P>

결핵균 자동염색기의 개발 및 평가

김수찬,강승일,김승철,황정호,김성녕,김영,송선대,조상래,김덕원,Kim, S. C.,Kang, S. I.,Kim, S. C.,Hwang, J. H.,Kim, S. Y.,Kim, Y.,Song, S. D.,Cho, S. N.,Kim, D. W. 대한의용생체공학회 2002 의공학회지 Vol.23 No.3

결핵을 진단하는 방법 중에서 신속하고 비교적 비용이 적게드는 방법은 객담을 통한 결핵균 도말 검사이다. 결핵균 도말 검사는 슬라이드에 도말한 환자의 객담을 가온 과정을 통해 고착시키고. acid-fast 염색방법을 통해 염색시킨 후 현미경으로 결핵균을 관찰하는 것이다. Acid-fast 염색방법은 크게 hot staining과 cold staining 방법 두 가지가 있으며, 우리나라에서는 염색 결과가 선명한 hot staining 방법인 Ziehl-Neelsen 방법을 주로 이용한다. 그러나, 기존의 결핵균 자동염색기는 가온 기능이 없어 환자의 객담을 슬라이드에 검사자가 고착을 시켜야 하고. 선명도도 낮은 문제점을 가지고 있다. 본 연구에서는 검사자의 인력 절감과 검사자 개인의 염색 능력에 따른 염색 정도의 변화를 줄이기 위해 가온이 가능한 결핵균 자동염색기를 개발하였다 개발된 염색기는 객담의 고착에서 염색 그리고 건조가지 전 과정이 자동으로 이루어진다. 염색 시간은 5개의 슬라이드를 고품질로 염색할 경우 21분이 소요되었다. 성능 평가를 위해 총 91개 객담을 대상으로 자동과 수동염색을 시행하여 일치율을 비교해 본 결과 75%로 통계적으로 유의한 차이를 보이지 않았다 (P>0.05). The detection of tubercle bacilli (TB) from sputum smear is one of the fast and inexpensive methods for diagnosis of tuberculosis. For this method. sputum smears are usually flexed by heating and stained by acid-fast staining method, and then examined under an optical microscope. Two Procedures are commonly used fur TB staining. One is hot staining and the other is cold staining method. The Ziehl-Neelsen method which is a hot staining method is widely used in Korea because its stained color is more vivid However, the conventional automated stainer has to fix the sputum smear on a slide manually and the stain is not so vivid because it has not heating function. In an effort to save labor and minimize variations in manual staining Procedure. we developed an automated stainer with heating function. The entire staining process is fully automated. from fixation to final washing and drying. With the automated methods, five slides can be flexed and stained in 21 minutes at consistent high quality We compared the concordance rate between the two methods for 91 sputum samples to validate the stain quality of the developed automated stainer. As the results, the concordant rate between the two methods was 95% and there was no significant difference (p>0.05)

위점막에서 헤마톡살련 염색시간을 연장한 메이어헤마톡살린-에오신염색에 의한 Helicobacter pylori-likeorganism 검출률과 특수 염색법들과의 비교

제갈승주 ( Seung Joo Jekal ),차현희 ( Hyun Hee Cha ),김신무 ( Shin Moo Kim ) 대한임상검사과학회 1999 대한임상검사과학회지(KJCLS) Vol.31 No.2

Helicobacter pylori-like organism(HLO) is more easily visua1ized with specia1 stainings than with hematoxylin-eosin, but this is time-consumed, expensive to use the routine diagnostic workup. We performed to verify whether Mayer``s hematoxylin-eosin staining method with prolonged hematoxylin time compares favorably with other well established specia1 stainings in the detection of HLO in gastic biopsy. Fifty gastric biopsy specimens(48 from antrum, two from body) , routinely forma1in f1xed and paraff1n wax embedded, from gastritis patients were stained with modified Harris`` hematoxylin-eosin(m-Harris HE) and modified Mayer``s hematoxylin-eosin m-Mayer HE) with prolonged hematoxyrlin time(10min), Warthin-Starry, Genta, AgNOR and EI-Zimaity staining. The detection rate for HLO was examined by divide into three groups (mild, moderate, severe) according to the degree of inflammation in the biopsy specimens. Density of HLO was scored from 0 to 5 by Genta``s c1assification. Using Warthin-Starry as a standard, positivity for HLO was 52% (26/50). Relative sensitivity for Warthin-Starry staining was 100% with m-Mayer HE, 96% with Genta, 96% with EI-Zimaity, 92% with AgNOR, and 80% with m-Harris HE staining. Relative sensitivity of HLO density for Warthin-stany stainings was lower at low (grade 1) density(Genta, 96%; EI-Zimaity. 96%; AgNOR, 33%, routine Harris HE, 0%) than at high(grade 2 to 5) density except case with m-Mayer HE(100%). Detection rate and density of HLO also appeared a significantly high according to increase the degree of inflammation. m-Mayer HE staining has a sensitiviη comparable to thoes of Warthin-Starry and Genta staining and is simple, inexpensive compared with the other staining methods in detecting HLO. Therefore, it is vety useful and practica1 for identifying HW in routine gastric biopsy specimens.

TTA법을 이용한 색소 질환에서 티로시나제의 발현 양상

이영규 ( Yeong Kyu Lee ),김영훈 ( Young Hun Kim ),김기호 ( Ki Ho Kim ) 대한피부과학회 2008 大韓皮膚科學會誌 Vol.46 No.3

Background: Tyrosinase is the critical enzyme in melanin synthesis. DOPA staining has been used as a standard assay for detecting tyrosinase activity, but it exhibits several limitations. Tyramide based tyrosinase assay (TTA) is a simple and sensitive tyrosinase detecting method. Objective: This study aimed to compare the stainability of pigmentary disorders using TTA and DOPA staining. Methods: The subjects were composed of hyperpigmentary disorders (n=10), hypopigmentary disorders (n=7), and alopecia areata (n=7). The colocalization study of TTA and Mitf using immunofluorescence was performed on alopecia areata. DOPA staining was performed on all tissues to compare with TTA immunohistochemistry. Results: TTA positive cells were correlated with Mitf positive cells in the tissue of alopecia areata. In hyperpigmentary disoders, TTA was stronger than DOPA staining. TTA and DOPA staining didn`t observe the positive cells in lesion of vitiligo and piebaldism, but both showed the positive cells in normal skin. TTA staining showed positive cells in the transitional lesion of vitiligo but, DOPA staining did not. In alopecia areata, TTA positive cells were observed, but DOPA staining did not. Conclusion: TTA is more sensitive than DOPA staining in pigmentary disorders for detecting tyrosinase activity. (Korean J Dermatol 2008;46(3):325∼333)

Neutral red 염색을 이용한 피부사상균의 생육성 평가

박장규 ( Jang Kyu Park ),이우재 ( Woo Jae Lee ),서기범 ( Ki Beom Suhr ),이증훈 ( Jeung Hoon Lee ) 대한피부과학회 1996 大韓皮膚科學會誌 Vol.34 No.1

Background: Microsopic examination of potassium hydroxide(KOH) preparation and fungus culture is required for the diagnosis of fungal infection. Sometimes dermatophytes fail to grow on culture medium, although these are observed on microscopic examination of KOH preparation. Recently this discrepancy between microscopic examination and fungus culture can be explained by the hyphothesis that some of the fungal elements are non-viable. Object: This study was made to evaluate the viability of dermatophytes using neutral red(NR) staining for the explaination of discrepancies between microscopic examination and fungus culture. Methods : After identification of fungus by culture from dermatophytic lesion the hyphae was collected for this study. In order to confirm whether the NR staining is suitable for check the viability of hypae or not, we designed to prepare the preparations of hyphaes by 2 ways. One was killed hyphae by autoclave, the other was kept as viable hyphae. And then we compared the stainability of NR staining and autoradiographic study using H-thymidine. And we compared the results of NR staining and the subsequent culture using the scales which were collected from the lesions 30 dermatophytic patients. Results : The structure inside of cell wall of hyphae stained red color only in case of viable hyphae preparations, but not stained in killed hyphae preparation. Autoradiographic study using H-thymidine confirmed that grain-positive cells(viable cells) were stained with NR, whereas grain negative cells (non viable cells) were not stained. Among the 30 cases with dermatophytosis 27(90.0%) cases showed NR positive and 14(46.6%) cases showed culture-positive. Except the tinea unguium cases which have shown low culture positive rate, 9(75.0%) cases of the 12 NR positive samples were positive on culture. All 14 cases of the culture positive samples were positive on NR staining. And all 3 cases of the NR negative samples were negative on culture. Conclusion : NR staining can be a useful method for the evaluation of viability of the fungal elements. (Kor J Dermatol 1996;34(1): 122-126)

Programmed Cell Death in Placenta

Kim, Yong-Beom,Kim, Jeong-Kyu,Hong, Seung-Hwa,Won, Choong-Hee,Lee, Seok-Jae,Hong, Chang-Soo,Ji, Ill-Woon,Jeong, Eun-Hwan,Kim, Hak-Soon 충북대학교 의학연구소 2001 忠北醫大學術誌 Vol.11 No.2

연구목적: 인간과 토끼의 태반에서 나타나는 세포고사(programmed cell death)의 양상을 알아보고자 하였다. 대상 및 방법 : 임신 17주에서 41주의 21명의 산모에서 얻어진 조직학적인 병변이 없는 태반과 임신 10일, 19일, 20일 그리고 27일의 토끼에서 얻어진 태반을 대상으로 연구하였다. 인간의 태반에서는 bcl-2의 발현양상을 면역조직화학염색과 immunoblotting analysis를 이용하여 검사하였고 세포고사가 나타나는 양상을 알아보기 위하여 TUNEL 염색과 agarose gelelectrophoresis of genomic DNA를 시행하였다. 또한 토끼의 태반에서 나타나는 세포고사의 양상을 알아보기 위하여 H&I 염색과 TUNEL 염색을 시행하였다. 결과: 임신 32주 이전의 태반에서는 모두에서 bcl-2의 발현이 양성이었다. 세포고사는 태반의 모든 조직에서 발견되었고 특히 탈락막 세포에서 많이 발견되었으며 세포고사 지수는 평균 4.57%이었다. 그러나 임신 주수에 따른 세포고사 지수의 변화는 통계학적으로 유의하지 않았다. 토끼의 태반에서도 세포고사는 H&I 염색과 TUNEL 염색으로 쉽게 관찰할 수 있었고 인간에서와 마찬가지로 탈락막에서 가장 많이 발견되었다. 결론: 태반에서 나타나는 세포고사는 인간과 토끼에서 모두 정상적으로 나타나는 소견이었다. 태반의 기저층에 해당하는 탈락막에서 세포고사가 가장 많이 나타났고 임신이 진행될수록 세포고사가 많이 나타났다. Purpose : We investigated the patterns of the programmed cell death (PCD) throughout the gestation in human and rabbit placental tissues. Materials and Methods: A total of 21 human placentas of gestational ages ranging from 17 weeks to 41 weeks which were devoid of histopathologic abnormalities and four rabbit Placentas of gestational ages of day 10, 19, 20 and 27 were obtained We have examined the patterns of bcl-2 expression in human placentas using immunohistochemical staining and immunoblotting analysis. We have used TUNEL staining and agarose gel electrophoresis of genomic DNA to ensure the patterns of PCD in human placentas. We also analyzed the pattern of PCD in rabbit placentas using hematoxylin and eosin (H&E) staining and TUNEL staining. Results : All of the placentas before the gestational age of 32 weeks were definitely positive for bcl-2 expression. PCD was observed in all cellular compartments of placenta and intermediate trophoblasts and decidual cells showed higher Prevalence of PCD. Apoptotic index was ranged from 3.30% to 5.67% (mean 4.57%). However, significant difference in the frequency of PCD according to the gestational age could not be demonstrated. In rabbit placenta, patterns of PCD were easily identified with TUNEL staining and hematoxylin and eosin staining and placental floor and decidua basalis were, as was suggested from human placenta, the major sites where PCD took place. Conclusion: We have clearly demonstrated that PCD is a normal finding in the placenta and decidua of both human and rabbit. The placental floor and decidua, the site of separation of fetomaternal interphase during the parturition, are the major compartment undergoing PCD. And also we have identified that PCD occurs more frequently with advancing gestation.

Different Profiles of the Negatively Stained Citrus Canker Bacterium Xanthomonas citri pv. citri Depending on Culture Media and Heavy Metal Stains

Kim, Ki-Woo,Lee, In-Jung,Hyun, Jae-Wook,Lee, Yong-Hoon,Park, Eun-Woo The Korean Society of Plant Pathology 2010 Plant Pathology Journal Vol.26 No.1

Staining profiles and bacterial morphology were compared in Xanthomonas citri pv. citri by a transmission electron microscopy. Four types of negative staining regimes were employed depending on culture media and heavy metal stains. The bacterial cells grown on LB agar media often appeared clustered on the supporting film. Meanwhile, individual bacterial cells could be readily found on the preparations from LB broth media. Typical rod-shaped cells (ca. $1\;{\mu}m$ in length) and their flagella were observed in either 2% uranyl acetate (UA) or 2% neutralized potassium phosphotungstate (PTA) staining. The UA-stained bacteria often showed relatively intact cell morphology and rather positively stained cells with a thin electron-dense stain depth around bacteria. The PTA-stained bacteria were characterized by the wrinkled cell surface where the stain was entrapped in grooves. In addition, distinct electron-dense stain depth was evident around the PTA-stained preparations. Numerous fimbriae could be mostly observed from the PTA-stained preparations of the two culture media, but not from the UA-stained preparations.

Different Profiles of the Negatively Stained Citrus Canker Bacterium Xanthomonas citri pv. citri Depending on Culture Media and Heavy Metal Stains

김기우,이인정,현재욱,이용훈,박은우 한국식물병리학회 2010 Plant Pathology Journal Vol.26 No.1

Staining profiles and bacterial morphology were compared in Xanthomonas citri pv. citri by a transmission electron microscopy. Four types of negative staining regimes were employed depending on culture media and heavy metal stains. The bacterial cells grown on LB agar media often appeared clustered on the supporting film. Meanwhile, individual bacterial cells could be readily found on the preparations from LB broth media. Typical rod-shaped cells (ca. 1 μm in length) and their flagella were observed in either 2% uranyl acetate (UA) or 2% neutralized potassium phosphotungstate (PTA) staining. The UA-stained bacteria often showed relatively intact cell morphology and rather positively stained cells with a thin electron-dense stain depth around bacteria. The PTA-stained bacteria were characterized by the wrinkled cell surface where the stain was entrapped in grooves. In addition, distinct electron-dense stain depth was evident around the PTA-stained preparations. Numerous fimbriae could be mostly observed from the PTA-stained preparations of the two culture media, but not from the UA-stained preparations.

Counterion-dye staining method for DNA in agarose gels using crystal violet and methyl orange

Yang, Yong Il,Jung, Da Woon,Bai, Dong Gyu,Yoo, Gyurng Soo,Choi, Jung Kap 전남대학교 약품개발연구소 2001 약품개발연구지 Vol.10 No.-

Sensitive and safe methods for visualization of DNA in agarose gels are described. 0.001 % crystal violet dissolved in distilled water was used for DNA staining on agarose gels and it could detect as little as 16 ng of DNA (3 kb, pGem-7Zf/EcoRl) without destaining procedure. The detection limit is four times lower than that of ethidium bromide. To improve the sensitivity, we studied a counterion-dye staining method using methyl orange as a counterion-dye which contributes to reduce excessive background staining by crystal violet. Dye concentration, pH of staining solution, mixing molar ratio of two dyes, and staining times were optimized for the counterion-dye staining. By the staining with a mixed solution of 0.0025% crystal violet and 0.0005% methyl orange in distilled water, 8 ng of the 3 kb DNA in an agarose gel was detected within 30 min.