Tuberculosis is a potentially deadly infectious disease caused by the Mycobacterium tuberculosis (M. tuberculosis). Tuberculosis is diagnosed by proving the M. tuberculosis in sputum samples based on the results of acid-resistant staining, culture, and nucleic acid amplification tests. However, there is a report that the detection rate of M. tuberculosis is low in acid-resistant staining using tissue specimens. It has been suspected that the cause is a potential loss of acid resistance by the organic solvents used for tissue specimen preparation. Therefore, this study was pursued to find out if Gram staining and fluorescent staining in addition to acid-resistant staining would be helpful in diagnosing tuberculosis. We used four tissue (lung, small intestine, large intestine, and lymph node) samples with chronic granulomatous inflammation observed in HE staining and positive results in real-time PCR. These detection rates and staining properties were investigated through microscopic examination using the Ziehl-Neelsen, Gram, and Auramin rhodamine staining. In this studies, M. tuberculosis were observed by Ziehl-Neelsen, Gram, and Auramin rhodamine staining in all four samples. In the evaluation of clinical microbiology proficiency testing (CMPT), the Ziehl-Neelsen and Gram staining were the same result, but the Auramin rhodamine staining was relatively low. These data indicated that Gram staining is useful for detecting M. tuberculosis in formalin-fixed tissue specimens. Therefore, if the Ziehl-Neelsen and Gram staining are combined as the M. tuberculosis staining method in tissue specimens, a better direction may be provided for tuberculosis diagnosis.

The acrosome cap allows sperm to penetrate the egg membrane and produce male pronuclei within female chicken eggs, facilitating successful fertilization. Given this, it is important to establish practical methods for evaluating the integrity of the acrosome cap and thus the quality of the rooster’s sperm. There are several established methods for evaluating the acrosomes of mammalian sperm, but none of these methods are suitable for evaluating the acrosome status of rooster spermatozoa. Therefore, a simplified method for evaluating the rooster acrosome is needed. Here we evaluated the usefulness of CBB (coomassie brilliant blue) staining of the acrosome at concentrations of 0.04%, 0.08%, and 0.3% CBB solutions. Our data revealed a clear staining pattern for intact acrosome caps at 0.04% and 0.08% CBB but not at 0.3% CBB. This protocol revealed differences in acrosome integrity between fresh and frozen rooster sperm smears suggesting that CBB staining may facilitate easier semen evaluation in roosters. This protocol allows for the accurate differential staining of acrosome cap in rooster spermatozoa.

Mitochondrion is an organelle for regulating calcium (Ca2+) homeostasis. Mitochondrial Ca2+ plays important roles on oocyte maturation, fertilization and embryonic development for ATP production. Low quality oocytes have mitochondrial dysfunction, which lead to overloaded Ca2+ in mitochondria. Recently, Rhod-2 is well known as a mitochondrial derived Ca2+ indicator. However, the changes of Rhod-2 in matured or fertilized porcine oocytes have not been reported. Therefore, the aim of study was to identify the effects of mitochondrial Ca2+ using Rhod-2 on quality assessment of matured oocyte and zygotes in pigs. Thus, we classified two groups (group 1: G1, compact COCs and group 2: G2, uncompact COCs) according to differences of cumulus cells amount and cytoplasm morphology in germinal vesicle (GV) stage of porcine COCs. Therefore, we investigated number of Rhod-2 spots in matured and fertilized oocytes from G1 and G2 groups. The Rhod-2 spot numbers were separated into four parts; n<10, 10≤ n < 20, 20 ≤ n < 30, and 30 < n. The Rhod-2 spots number of G2 group had greater than G1 group in part of 20 ≤ n. Additionally, we investigate mean number of Rhod-2 spots from G1 and G2 groups in matured and fertilized oocytes. As a result, we confirmed that average number of Rhod-2 spots in G2 group increased than that of G2 group. Finally, we also measured the Rhod-2 intensity in matured and fertilized oocytes of G1 and G2 groups. Interestingly, the Rhod-2 intensity in G2 group was higher than that of G1 group. (oocyte: p < 0.001 and fertilized oocyte: p < 0.05). These results demonstrated that changes in Rhod-2 spots and intensity were increased in low quality of matured and fertilized oocytes. Therefore, our results suggest that the differences in mitochondrial calcium level are associated with morphological quality of porcine COCs.

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study.

The brilliant cresyl blue (BCB) has been used to select the developmental competent oocytes in pigs, goats and cows. Growing oocytes have a higher level of active glucose-6-phosphate dehydrogenase(G6PDH) compare to mature oocytes and are rarely stained compared to mature oocytes, because G6PDH converts BCB to colorless. First polar body extrusion regard as a guideline of meoisis completion. Selection of polar body extrude oocyte is more developmental competent to blastocyst than unselected. This study was conducted to compare the BCB test to the polar body extrusion on selection of developmental competent porcine oocytes for the production of blastocyst. Cumulus-Oocytes complex were exposed to 26uM BCB stain diluted in NCSU-23 for 90 min. There was no significant difference embryo development to blastocysts between BCB treated and not treated(19.58±1.99 vs 18.75±2.27 %), which means there was no detrimental effect of BCB exposure to oocytes. Normal fertilization is not differed among treatment groups from 70.0 to 78.4% development to blastocyst, beside polyspermy did not. To compare two different selection methods, BCB test and polar body extrusion, evaluate the developmental competent of IVP embryos. BCB+PB+(blue stained and polar body extruded, 20.71±0.45%) and BCB－PB+(colorless and polar body extruded, 20.04±1.29%) groups are significantly (p<0.05) higher developed than those of BCB+PB－(blue stained and no polar body, 13.24±0.73%) and BCB－PB－(colorless and no poladbody, 7.25±0.77%). These results showed that selection of polar body extruded oocytes method is more efficient than that of BCB test.

30년 전부터 보고되기 시작한 3-9시 방향 스태이닝온 하드 콘빽트 핸즈훌 확용하 는 모든 환자률에게 가장 쉽게 나타날 수 있는 중상 중의 하나이다. 환자들이 주로 호소하는 증상으로는 렌즈 착용 시간과 비례한 건조감， 껄끄러움， 핸즈에 대한 민갑 함 둥이다. 각막 주변부 건조중은 불안정한 누액파 함째 3-9시 방향 스태이닝의 원 인이다. 각막 건조중은 여러 가지 요인이 상호 복합척으로 작용하여 이루어지기 때문 에 주원인에 대해 정확한 분석융 하여야만 한다.3-9시 방향 스태이닝의 문제롤 해 결하기 위해서는 주변부 각막건조에 기여하는 요인올 찾는 것이 중요하다. 본 조사에서는 중상， 원인， 그리고 해결 방법을 체계적으로 앓아보았다.

The object of this study was to find simple and effective methods for the speculation of vitality and scorsome status of bovine spermatozoa. The eosin-nigrosin staining, trypan blue staining, and naphthol yellow S-erythrosin B staining was ofter used for the speculation of vitality and/or acrosome status of bovine spermatozoa, respectively. This study has shown that the combined trypan blue-naphthol yellow S-erythrosin B staining is more accurate and effective for the examination of acrosome status and vitality of bovine spermatozoa.

Genome sequencing researches for considerable numbers of crops and wild plants are being developed. Cytogenetic researches according to chromosome number and size are essential to confirm and comprehend ploidy level and genome size before genome sequencing project is actually conducted. Cytogenetic researches on six food crop plants were carried out by DAPI staining and fluorescence in situ hybridization (FISH) method. Fagopyrum esculentum Moench showed 2n=2x=16, each chromosome length of 1.42㎛ to 1.77㎛, total chromosome length of 13.31㎛, and karyotypic formula of 2n=8m; Phaseolus angularis W.F. Wight, 2n=2x=22, 2.01㎛ to 3.84㎛, total 28.03㎛, 2n=9m+2sm, Perilla frutescens var. japonica Hara, 2n=2x=40, 1.73㎛ to 2.76㎛, total 44.36㎛, 2n=5m+13sm+2st. Chromosome sizes of the other three species such as, Panicum miliaceum L., 2n=2x=36, total chromosome length of 30.83㎛, Sesamum indicum L., 2n=2x=26, 27.39㎛, lpomoea batatas L., 2n=2x=30, total 33.51㎛ were too small for each chromosome type to be identified and analyzed. The result of FISH analysis using 5S and 45S rDNA probe showed species-specific chromosome locations in the genome. These preliminary analyses were carried out to decide which food crop to prioritize for genome sequencing. This work was supported by the “Cooperative Research Program for Agriculture Science & Technology Development (No.PJ009837), Rural Development Administration, Republic of Korea.

충치는 사람의 구강질환 중 가장 흔한 질환으로 Streptococcus mutans (S. mutans)균이 초기 충치를 형성하는데 매우 중요한 역할을 담당한다. Porphyromonas gingivalis (P. gingivalis)는 대표적인 구취 유발균으로 구취 형성에 중요한 휘발성 황화합물을 생성하는데 관여한다. 치주질환은 치은결체조직과 치조골의 파괴를 유발하여 치아의 상실을 초래할 수 있는 만성 염증성 질환으로 Prevotella intermedia (P. intermedia)가 원인균이다. 이번 연구에서는 cetylpyridinium chloride (CPC), sodium fluoride (NaF), 녹차 추출액, 솔잎 추출액을 유효성분으로 하는 마우스워시 제품을 사용하여 S. mutans 균을 포함, 구강질환 균으로 널리 알려진 P. gingivalis, P. intermedia 대해 항균 효과를 확인하고자 하였다. 그 결과 시험군의 경우 S. mutans, P. gingivalis 에 대해 30 s 내에 4.00 Log, 4.68 Log의 사멸력을 확인하였고, P. intermedia의 경우 30 s 2.40 Log, 60 s 2.70 Log 사멸력을 확인하였다. 또한 Dentocult SM Strip mutans (SM Strip) 염색방법을 적용하여 S. mutans 균의 감소여부를 시각적 자료로 쉽게 확인할 수 있었다. 이와 같은 결과를 통해 CPC, NaF, 녹차 추출액, 솔잎 추출액을 포함한 마우스워시 제품은 구강균 사멸을 통해 충치 및 구취와 같은 구강질환 예방에 효과가 있을 것으로 기대한다.

화장품에 배합되는 원료 중에 피부 모공을 막아 여드름의 초기단계인 면포를 유발함으로써 염증성 여드름으로 악화시키는 사례가 보고되고 있다. 본 연구는 ‘여드름 피부에 사용이 적합한’ 면포 비유발성 화장품의객관적인 평가 기준을 마련하고자 하였다. 외국 임상기관에서 실시하고 있는 non-comedogenic test를 조사하고 등 상부 반복 폐쇄 첩포를 통해 면포 유발 평가방법을 확립하고자 연구를 수행하였다. 또한 안면부 사용성시험을 동일 피험자에게 추가로 진행하였다. 등 상부 반복 폐쇄 첩포를 통해 면포를 채취하여 분석한 결과 시험시료로 사용한 보습제와 자외선 차단제가 면포를 유발하지 않음을 확인할 수 있었고, 동일한 시험제품을 안면부에 사용하여 얻은 면포 유발 결과와 Global acne grading system (GAGS)의 여드름 육안평가 결과는 상관관계가 없었다. 또한 사진 판독을 통해 분석하는 면포와 모낭의 구별을 용이하게 하기 위해 면포 채취 표본에 Oilred O staining을 실시하였다. 염색을 한 경우의 표본이 염색을 하지 않은 표본과 비교하여 결과 간 높은 일치도를 확인하였다. 본 연구에서는 Oil red O staining을 통해 객관성과 신뢰성을 증진시킨 새로운 버전의 화장품면포 비유발성 평가법을 확립하였다.

Conventional staining and fluorescence in situ hybridization (FISH) karyotypes of the non-genetically modified (GM) parental rice line, 'Nakdong' (Oryza sativa L. japonica), and its four GM rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1), and LS28 × Cry1Ac1 (events L/C1-1-3-1 and L/C1-3-1-1) were analyzed using 5S and 45S rDNAs as probes. Both parental and transgenic lines were diploids (2n=24) with one satellite chromosome pair. The lengths of the prometaphase chromosomes ranged from 1.50 to 6.30 μm. Four submetacentric and eight metacentric pairs comprised the karyotype of 'Nakdong' and its four GM lines. One pair of 5S rDNA signals was detected near the centromeric region of chromosome g in both the parental and transgenic lines. The 45S rDNA signals were detected on the secondary constrictions of the satellite chromosome pair in both the parental and transgenic lines. There was no significant difference in chromosome size, length, and composition between 'Nakdong' and its four GM lines. This research was conducted as a preliminary study for chromosomal detection of transgenes in GM rice lines and would be useful for their breeding programs.

교배효율의 증대를 위해서는 정상화분의 확보가 선행되어야하며 화분의 활성을 파악할 필요가 있다. 본 연구에서는 화분의 활성을 확인하기 위해 여러 가지 염색액을 사용하여 광학현미경하에서 관찰하였다. Aceto-carmine과 Alexander's 염색액을 사용한 실험에서 비슷한 정상화분율을 얻을 수 있었고 FCR염색으로 관찰한 결과 살아있는 화분만 정확히 선별해 주기에 가장 낮은 정상화분율을 얻을 수 있었다. TB는 화분의 상태에 따라 달랐으나 Aceto-carmine과 Alexander's 염색액을 사용한 실험에서의 결과와 비슷한 양상을 보였다. 화분 관찰의 경험이 별로 없는 실험자도 쉽게 접근할 수 있는 염색액은 Alexander's 이었다. 정확한 화분의 활성을 조사하는 데에는 FCR이 필수이다. 형광현미경 장치 없이 정상과 비정상화분의 구별이 용이한 Alexander's 염색액으로 관찰한 자료를 바탕으로 살아있는 정상화분율의 예견이 가능하나 정확한 임성을 확인하려면 추가로 FCR test를 해야 한다.