• Applied Microscopy
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• 제42권2호
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• pp.111-114
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• 2012

The scanning electron microscopy is an ideal technique for examining plant surface at high resolution. Most hydrate samples, however, must be fix and dehydrate for observation in the scanning electron microscope. Because the microscopes operate under high vacuum, most specimens, especially biological samples, cannot withstand water removal by the vacuum system without morphological distortion. Cryo-techniques can observe in their original morphology and structure without various artifacts from conventional sample preparation. Rapid cooling is the method of choice for preparing plant samples for scanning electron microscopy in a defined physiological state. As one of cryo-technique, high-pressure freezing allows for fixation of native non-pretreated samples up to $200{\mu}M$ thick and 2 mm wide with minimal or no ice crystal damage for the freezing procedure. In this study, we could design to optimize structural preservation and imaging by comparing cryo-SEM and convention SEM preparation, and observe a fine, well preserved Arabidopsis stem's inner ultrastructure using HPF and cryo-SEM. These results would suggest a useful method of cryo-preparation and cryo-SEM for plant tissues, especially intratubule and vacuole rich structure.

• Clinical and Experimental Reproductive Medicine
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• 제17권1호
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• pp.81-85
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• 1990

We cryopreserved mouse morulae by a simple ultra-rapid method of freezing embryos directly in $LN_2$ after holding 2min in a $LN_2$ vapor, and thawed them in $37^{\circ}C$ water bath. The time requirements for permeation and dehydration by 2.0 M glycerol and 0.2 M sucrose before freezing were studied. When the embryos were equilibrated for 10 min, the optimun post-thaw survival was obtained. Embryos those developed normally to blastocyst after in vitro culture for over 24hrs were regarded as survival ones. Two experiments to assess post-thaw survival following predehydration in various mixtures of glycerol and sucrose were also accomplished. When sucrose was held constant (0.2 M) and glycerol concentration varied (1.5-3.5 M), post-thaw survival was best (78.0%) in 3.0 M glycerol. When glycerol was held constant (3.0M) and sucrose concentration varied (0.0-1.0M), optimun post-thaw survival (78.0%) was found in 0.2 M sucrose.

• 한국농업기계학회:학술대회논문집
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• 한국농업기계학회 1993년도 Proceedings of International Conference for Agricultural Machinery and Process Engineering
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• pp.443-452
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• 1993

Direct freezing tests of soybean seed by liquid nitrogen were carried out at various moisture contents and the following important conclusions were drawn from the results of temperature measurements of soybean seed and photographs of bubbles generated on its surface : 1) Assuming that the temperature gradient in a soybean seed is negligible because of its small seed size and the freezing ratio is followed the Heiss's formula, and a differential equation based on the heat energy balance was introduced . The equation was easily solved by the Runge-Kutta-Gill method and the predicted values of the temperature were in good agreement with the observed data. 2) The photographs of bubble generation during freezing showed the boiling mode was nucleate, and then the most suitable formula on the nucleate boiling heat transfer was introduced from many formulate proposed up to now by fitting the calculated values based on the formula to the observed data. The formula used for the predict on of the seed temperature was as follows: $\frac{{\partial}T_s}{\partial\theta}\;=\;-\frac{{\alpha}(T_s\;-\;T_L)^{3.3}}{W(C_s\;-\;\frac{{\delta}m(CT_s\;+\;{\sigma})}{T_s^2})}$ where C = difference of the specific heat between pure ice and water m=moisture content of soybean seed $T_s$ = seed temperature $T_L$ = Temperature of liquid nitrogen W = mass of soybean seed $\alpha$ = proportional constant $\delta$ = constant depends on variety or the type of seed $\theta$ = time $\sigma$ = latent heat of melting of pure ice This study will give important information in the hydro-freezing technique by liquid nitrogen, available as a new technique of processing agricultural products in the near future.

• Journal of Applied Biological Chemistry
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• 제58권1호
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• pp.55-60
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• 2015

수출용 냉동딸기 제조 시 미생물학적 안전성 확보를 위한 수단으로 이산화염소수와 acetic acid 병합처리를 최적 비가열 전처리 기술로써 적용하여 -20, -70, $-196^{\circ}C$로 냉동한 후 미생물 수, 품질변화 및 관능평가를 조사하였다. 냉동방법 및 세척 처리에 따른 색도 차이는 나타나지 않았고, 비타민 C 함량은 $-70^{\circ}C$ 냉동에서 35.33 mg/100 g FW로 가장 대조구와 유사하였으며, drip loss도 $-70^{\circ}C$ 냉동이 14.39%로 가장 낮게 나타났다. 관능평가 역시 $-70^{\circ}C$ 냉동이 -20, $-196^{\circ}C$ 냉동보다 높은 점수를 받았으며, 세척처리는 비타민 C 함량, drip loss 및 관능평가에 큰 영향을 끼치지 않는 것으로 나타났다. 또한, 냉동딸기에 50 ppm 이산화염소수와 1% acetic acid를 병합 처리하여 냉동 후 미생물 수 변화를 측정한 결과, 냉동 전과 같이 병합처리 된 딸기 시료에서 미생물이 검출되지 않았다. 따라서 저장성이 높은 수출용 냉동딸기 생산을 위해서는 gas nitrogen convection chamber를 이용한 $-70^{\circ}C$에서의 급속냉동 처리가 보다 효과적인 냉동방법이며, 냉동 처리만으로는 미생물 제어가 어렵기 때문에 냉동 전 비가열 전처리를 통해서 냉동딸기의 미생물학적 안전성을 확보해야 한다고 판단된다.

• Asian-Australasian Journal of Animal Sciences
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• 제13권3호
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• pp.313-316
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• 2000

Three Holstein steers, fitted with ruminal and duodenal cannulas, were used in a replicated $3{\times}3$ Latin square design to determine the digestibility of dietary nitrogen in total digestive tract by three methods, 1) mobile nylon bag (MNB); 2) total fecal collection (TFC); and 3) washed fecal sample after freezing and thawing through a sieve with a pore size of $45{\mu}m$ (WFS). A basal diet of oaten hay-barley was supplemented with one of the following protein sources; soybean meal, fish meal or blood meal. Steers were fed at a level of 2% of body weight. The experimental diets were contained approximately 1.85% nitrogen. There were no differences (p>0.05) among the diets on DM, NDF and nitrogen disappearances, and the diet results were pooled to assess the methods. Total tract disappearances of dry matter and neutral detergent fiber were 61.6, 71.1 and 78.9 and 25.3, 63.2 and 64.6 for MNB, TFC and WFS methods, respectively. The lower digestibility of DM and NDF in the MNB method could be a result of low ruminal incubation time. The TFC method had the lower (p<0.05) determination of nitrogen disappearance in the total digestive tract than the MNB and WFS methods. On the other hand, nitrogen disappearance in the total digestive tract determined by the WFS technique was comparable to that in MNB technique, as there was no significant difference (p>0.05) between the methods. It is shown that the disappearance of dietary nitrogen in the total digestive tract could be estimated in the intact animals by using washed fecal sample prior to freezing and thawing.

• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.71-76
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• 1998

본 연구는 소 미성숙난자를 electron microscope (EM) grid와 동해제인 EFS30을 이용하여 초급속 동결하였을 때 정상적인 배 발달의 유도가능성 여부를 조사하고자 실시하였다. 동해제는 30% ethylene glycol, 18% ficoll, 0.5 M sucrose와 10% FBS 등이 PBS에 첨가되어 제작된 EFS30을 사용하였다. 난자 생존의 평가기준으로는 성숙, 수정 및 배발달을 조사하였다. 본 연구에서 얻어진 결과는 다음과 같다. 초급속동결-융해 후, 소 미성숙란의 생존율은 43.2%을 나타내었다. 동결-융해군의 체외 성숙 (84.1%)과 정상 자웅전핵 형성율 (57.5%)은 대조군의 결과 (92.5, 65.0%)와 비교하여 볼 때 유의한 차이는 없었다. 또한, 동결군의 체외수정 이후의 $\geq2$-세포기 형성 (65.0%)과 배반포형성율 (30.8%)도 대조군(73.7, 35.7%)의 결과와 유의한 차이를 나타내지 않았다. 따라서 소 미성숙난자는 EM grid와 EFS30 동결액을 이용한 초급속 동결방법에 의해 정상적인 배발달이 유도될 수 있다는 것을 알 수 있었다.

• Restorative Dentistry and Endodontics
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• 제35권4호
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• pp.285-294
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• 2010

본 연구의 목적은 흰 쥐의 상악 대구치를 발거한 후 치주인대세포를 $0^{\circ}C$/2 MPa고압-저온하에 1주간 보관시켜 MTT, WST-1 검색법을 이용하여 측정한 치주인대세포의 활성도를 저속 냉동법(No Additional Pressure, 2, 3 MPa), 급속 냉동법(No Additional Pressure, 2 MPa), $-5^{\circ}C$/90 MPa초고압 저온보존법과 비교하여 평가하는 것이다. 생후 4주된 암컷 Sprague-Dawley계 흰쥐의 상악 좌우 제1, 2대구치를 발거하여 각 군 당 12개의 쥐 치아를 MTT, WST-1 검색에 이용하였다. 실험군은 9개군으로 대조군은 즉시 발치군이며, 각각 3 MPa, 2 MPa, No Additional Pressure (NAP)의 압력을 가한 후 $4^{\circ}C$에서 $-35^{\circ}C$까지 $-0.5^{\circ}C$/min 속도로 서서히 냉동시킨 뒤 $-196^{\circ}C$에 냉동한 저속 냉동군, 발치 후 동해방지제 처리과정을 거쳐 각각 2 MPa, NP의 압력을 가한 후, $-196^{\circ}C$의 액화질소에 넣어 냉동한 급속 냉동군, 발치 후 각각 2MPa,NP의 압력을 가한 후, $0^{\circ}C$에 보관한 저온 보존군, $-5^{\circ}C$/90 MPa의 초고압 저온 보존군으로 나누었다. 보존액은 F medium을 사용했으며 동해방지제로 10% dimethylsulfoxide (DMSO)를 사용하였다. 치근면을 단위면적으로 표준화하기 위해 MTT, WST-1 측정값을 Eosin 염색 후 530 nm에서 측정한 흡광도 값으로 나누었다. 통계 분석을 위해 one way ANOVA를 시행하였으며 사후 검정으로는 Tukey HSD 방법을 사용하였고 결과는 다음과 같다. 1. MTT 검색법 및 WST-1 검색법 결과 $0^{\circ}C$/2 MPa 고압 저온 보존군이 즉시 발치군보다 세포 활성도가 낮았으나 통계적 유의차는 없었으며, 저속 압력 냉동군(NP, 2 MPa, 3 MPa)과, 급속 압력 냉동군(NP, 2 MPa), 저온보존군($0^{\circ}C$/NP), 초고압 저온 보존군($-5^{\circ}C$/90 MPa)보다 통계적으로 유의차있게 높은 세포 활성도를 나타내었다(p < 0.05). 2. MTT검색법 및 WST-1 검색법 결과 $-5^{\circ}C$/90 MPa 초고압 저온 보존군이 가장 낮은 세포 활성도를 나타내었으며, MTT 검사 결과에서는 모든 군에 대해 통계적으로 유의성 있는 결과를 보였다(p < 0.05). 위의 결과를 통해, $0^{\circ}C$/2 MPa (20기압)의 고압-저온 보존법이 다른 급속 냉동 보관법(2 MPa, NAP)이나 저속냉동보관법(3, 2 MPa, NAP), $-5^{\circ}C$/90 MPa 초고압 저온 보존법에 비해 우수한 쥐 치아의 치주인대세포의 활성도를 보여 차후 치아의 재이식시 치아보관을 위한 방법으로의 가능성을 제시하였다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.6-6
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• 2001

Oocyte freezing has become a prevalent source for related reproductive technologies. This study was carried out to evaluate viability of post-thawed bovine oocyte injected DTT-treated sperm following by two different activation stimuli (Group 1, 5 M ionomycin, 5 min ＋ CR1aa, 3 h . 1.9 mM dimetylaminopurine (DMP), 3 h; group 2, ionomycin ＋ 10 $\mu\textrm{g}$/$m\ell$ cycloheximide(CHX), 5h). The techniques of ultra-rapid freezing used in this study were essentially similar to those of described by Vajta et al (Theriogenology 1999; 52:939-948), Denuded oocytes at 22 h of culture were exposed to cryoprotectant (3.2 M Ethylene glycol, 2.36 M DMSO, 0.6 M sucrose), and followed by freezing in electron microscopic grid. After thawing the oocytes were transferred back into the drop of maturation medium and cultured for additional 2 h before being subjected to ICSI. All eggs were then cultured in CRlaa medium, and transferred into M199+10% FCS on day 4. The culture was maintained until day 9. In Experiment 1, frozen-ICSI eggs were compared on development into blastocyst to those of unfrozen and IVF control. Those eggs were activated with the method of group 2. A higher proportion of unfrozen-ICSI and IVF eggs developed into cleavage and blastocysts than of frozen-ICSI eggs (65% and 13%; 71% and 23% vs. 39% and 8%; P<0.05). In Experiment 2, development and ploidy of embryos made from group 1 were compared to those from group 2. Between groups there did not differ on the rates of development, however, chromosomal abnormality in group 1 was significantly higher than in group 2 (49% vs. 30%; P<0.05). The present result suggests that frozen bovine oocytes can be used for ICSI.

• 한국콘크리트학회:학술대회논문집
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• 한국콘크리트학회 1997년도 가을 학술발표회 논문집
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• pp.313-318
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• 1997

Air voids existed in hardened concrete have an important influence on concrete deterioration such as carbonation, freezing and thawing, and corrosion of embedded steel in concrete. Therefore it is very significant to investigate the pore structure of system (size, number and continuity of air voids) to solve the reason caused concrete deterioration. The purpose of this study is to develop th standard method of measuring air voids which affect properties in hardened concrete using image analyzing system. This paper presents the settlement of rapid and exact experimental method which extracts fine bubbles, calculates the number of air voids, and determines air-voids distributions using image analyzing system with computer.

• 한국수정란이식학회지
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• 제29권3호
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• pp.241-248
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• 2014

본 연구에서 생쥐 초기 배아를 이용하여 동결 보존된 배아의 생존율 또는 발달율에 영향을 미치는 요인들의 상관관계를 알아본 결과, 배아의 발달 단계에 따른 동결 - 해동 후 생존율에 있어서는 2세포기 배아가 4~8세포기 배아보다 유의하게 높았으나(p<0.01), 배 발달율에 있어서는 4~8세포기 배아가 유의하게(p<0.01), 높아 생존율과 배 발달율과는 상관관계가 없는 것으로 사료된다. 또한, 동결 보호제에 따른 배아의 생존율에 있어서는 2, 4~8세포기 배아 모두 DMSO에서 유의하게 높았으나(p<0.01), 배 발달율에 있어서는 EG가 DMSO에 비해 더 유의하게 높은 성적을 나타내어(p<0.01, p<0.05) 동해로 인한 상해가 적은 것으로 생각되어 2, 4~8세포기 배아에서 EG가 더 효과적인 동결 보호제로 사료되며, 동결 프로그램으로는 완만 동결 - 급속 해동법이 더 우수한 프로그램으로 보인다.