| 產品名稱： | ATCC培養基712；含補充劑的PYG培養基 |
|---|---|
| 英文名稱： | ATCC medium 712；PYG w/ Additives |
| 培養基類型： | 非選擇性培養基 |
| 級別： | for microbiology |
| 品牌名稱： | ELITE-MEDIA（艾禮培養基） |
| 產品目錄號： | M870-01、M870-02 |
| 產品規格： | 5x500ml（無菌液體）、20個平板/包（無菌即用固體培養基） |
| 產品外觀： | 米色至麥秸色均一粉末。 |
| 顏色與澄清度： | 中等琥珀色溶液/凝膠。 |
| 保存條件： | 密封，避光，2-25°C保存。 |
| 注意事項： | 避免攝入、吸入、皮膚接觸。 |
| 相關產品： | -- |
產品描述：
ATCC Medium 712：含補充劑的PYG培養基(PYG w/ Additives)用于培養卡氏棘阿米巴(Acanthamoeba castellanii ATCC PRA¬107)。配方與配制方法
| Proteose Peptone | 20.0 g |
| Yeast Extract | 1.0 g |
1.用900ml DI Water 溶解培養基基礎21.0g，如果配制固體培養基，加入15g瓊脂。
2.依次加入下表中的貯存溶液，以避免沉淀生成。
3.檢查pH值是否為6.5。
4.121℃高壓蒸汽滅菌15min。
5.待冷卻至55℃左右時，加入50ml 過濾除菌的2 M葡萄糖溶液（18g/50ml）。
貯存溶液：
| 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 |
培養方法：
生長條件：25.0°C，持續無菌培養。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.
培養間隔時間：每月。
培養物維護：
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.
冷凍保存：
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.