連結的部分稍晚再貼...因為我ie很怪... pcman跟ie合開必當@@ 電顯 第二題 2. Cryobiology is a recently well-developed integrated field in biological and physical sciences. Would you please mention principle method, application and limitations of different cytotechniques, e. q. cryopreservation of sperm and/ or plant calli, freeze-drying, freeze-substitution, freeze-fracture, freeze-et ching and cryoSEM. Ans; Cryopreservation of sperm --method 1. Blood test to screen for infectious diseases such as HIV (the AIDS virus) , hepatitis and syphilis. 2. Semen collection 3. The sperm will be mixed with a solution to reduce freezing damage and then frozen in colour-coded, labeled plastic straws and stored in liquid nitrog en at -196℃. 4. Thawed in 40℃ water bath. --application Homologous and donor insemination, cryopreservation prior to surgical infertil ity treatment, intraoperative cryopreservation, postoperative cryopreservation , cryopreservation prior to treatment for malignancies and nonmalignant diseas es, as well as premortem and postmortem cryopreservation. --limitation There is concern about the transfer of genetically damaged sperm after cryopre servation. There is no guarantee that the sperm will survive thawing. There is also no guarantee that the thawed sperm will be fertile or that they will giv e rise to a conception if used with technologies such as artificial inseminati on, in vitro fertilization (IVF) and sperm microinjection (ICSI). Cryopreservation of plant calli --method 1. Calli samples were taken after subculture 2. Cryoprotectants were used: (1). 10% DM-SO + 0.5 mol/L sorbitol; (2). 7.5% DMSO +5% glycerin +5% sucrose. 3. The specimens were cooled at a rate of l℃/min from 0℃, kept -40℃ for 2 h, put into liquid nitrogen(-196℃) 4. Thawed in 40℃ water bath. --application --limitation Freeze-drying --method 1. Deep-freeze 2. Sublimation: A vacuum is applied to pull all the water out in the form of water vapor. 3. Slowly returned to room temperature. --application Preservation of materials, e. q. tissues for transplant, fresh flowers, fr uits, and vegetables that maintain the natural shape and vibrant color of the botanical --limitation Freeze-drying whole organs (or bodies) would be much more difficult and ti me-consuming, though, so it's not clear that this approach will ever be a usef ul alternative to cryonic suspension. Freeze-substitution --method 1. Cryoprotection and high-pressure freezing 2. Substituted frozen water with acetone containing some additives at -80℃ to -95℃ 3. Warm up to r. t. 4. Embedding --application Preservation of cell structure, immunolabeling, and embedding specimens in to resin --limitation Freeze-fracture --method 1. Cryoprotection 2. Quick freeze in liquid nitrogen 3. The frozen specimen is rapidly transferred to a precooled stage (-150℃) 4. Chamber pumped to high vacumn 5. The stage is warmed to -100℃ and the tissue fractured by razor blade 6. Platinum is evaporated at 45。 relative to the specimen plane 7. Carbon is evaporated directly above 8. The chamber is brought to r. t. and pressure 9. The thawed tissue is digest in 5% sodium hypochloride 10. The replica is moved to an additional chlorine bleach change and then through 2 or 3 water washes 11. The replica is put on a meshed grid and dried 12. The replica is examined under a TEM --application Examine membrane faces --limitation Freeze-etching --method 1. Cryoprotected 2. Freeze 3. Fractured directly over the specimen 4. Warm up to -100℃ and remove the ice by sublimation at a rate of 2 to 3 nm per second --application Examine actual membrane surface --limitation 1. Poor freezing and accidental warming of tissue may lead to ice crystal formation, causing deformation of tissue structure. 2. The replica is fragile and brittle and may fragment CryoSEM 尋找中......@~@ --

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