| 产品名称： | ATCC培养基712；含补充剂的PYG培养基 |
|---|---|
| 英文名称： | ATCC medium 712；PYG w/ Additives |
| 培养基类型： | 非选择性培养基 |
| 级别： | for microbiology |
| 品牌名称： | ELITE-MEDIA（艾礼培养基） |
| 产品目录号： | M870-01、M870-02 |
| 产品规格： | 5x500ml（无菌液体）、20个平板/包（无菌即用固体培养基） |
| 产品外观： | 米色至麦秸色均一粉末。 |
| 颜色与澄清度： | 中等琥珀色溶液/凝胶。 |
| 保存条件： | 密封，避光，2-25°C保存。 |
| 注意事项： | 避免摄入、吸入、皮肤接触。 |
| 相关产品： | -- |
产品描述：
ATCC Medium 712：含补充剂的PYG培养基(PYG w/ Additives)用于培养卡氏棘阿米巴(Acanthamoeba castellanii ATCC PRA¬107)。配方与配制方法
| Proteose Peptone | 20.0 g |
| Yeast Extract | 1.0 g |
1.用900ml DI Water 溶解培养基基础21.0g，如果配制固体培养基，加入15g琼脂。
2.依次加入下表中的贮存溶液，以避免沉淀生成。
3.检查pH值是否为6.5。
4.121℃高压蒸汽灭菌15min。
5.待冷却至55℃左右时，加入50ml 过滤除菌的2 M葡萄糖溶液（18g/50ml）。
贮存溶液：
| 0.05M CaCl2 | 8.0 ml |
| 0.4 M MgSO4 x 7H2O | 10.0 ml |
| 0.25 M Na2HPO4 x 7H2O | 10.0 ml |
| 0.25 M KH2PO4 | 10.0 ml |
| Na Citrate x 2H2O | 1.0 g |
| 0.005 M Fe(NH4)2(SO4)2 x 6H2O | 10.0 ml |
培养方法：
生长条件：25.0°C，持续无菌培养。Open a freezedried vial. Aseptically add 0.5 ml of ice cold medium containing 12% (w/v) sucrose to the freezedried inner shell vial. Once the culture is completely rehydrated, aseptically add 1 ml of ATCC medium 712 and distribute to a 16 X 125 mm plastic screwcapped test tube or a T25 tissue culture flask containing 5.0 ml of the same medium. Incubate the test tube culture horizontally with the cap on tight. Trophozoites should be evident in 15 days.
培养间隔时间：每月。
培养物维护：
1.When the culture is at or near peak density, vigorously agitate the culture.2.Transfer approximately 0.25 ml to a fresh tube or flask containing 5 ml of fresh ATCC medium 712.
3.Screw the caps on tightly and incubate at 25°C (incubate test tubes at a 15° horizontal slant).
4.The amoebae will form an almost continuous sheet of cells on the bottom surface of the flask or test tube.
Repeat steps 13 at 1014 d intervals.
冷冻保存：
在液氮中保存。1.To achieve the best results set up cultures with several different inocula (e.g. 0.25 ml, 0.5 ml, 1.0 ml). Harvest cultures and pool when the culture that received the lowest inoculum is at or near peak density.
2.If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 106 and 2 x 107cysts/ml with fresh medium. If the concentration is too low, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
3.While cells are centrifuging prepare a 15% (v/v) solution of sterile DMSO as follows:Add the required volume of DMSO to a glass screwcapped test tube and place it in an ice bath.Allow the DMSO to solidify. Add the required volume of refrigerated medium.Dissolve the DMSO by inverting the tube several times.
*NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
4.Mix the cell preparation and the DMSO in equal portions. Thus,the final concentration will be between 106 and 107 cells/ml and 7.5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
5.Dispense in 0.5 ml aliquots into 1.0 2.0 ml sterile plastic screwcapped cryules (special plastic vials for cryopreservation).
6.Place the vials in a controlled rate freezing unit. From room temperature cool at 1°C/min to 40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at 1°C/min through the heat of fusion. At 40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at 80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.(The cooling rate in this apparatus is approximately 1°C/min.)
7.The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
8.To establish a culture from the frozen state place an ampule in a water bath set at 35°C (23 min). Immerse the vial just sufficient to cover the frozen material. Do not agitate the vial.
9.Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 5 ml of fresh ATCC medium 712 in a T25 tissue culture flask or plastic 16 x 125 mm screwcapped test tube. Incubate at 25°C.
